Bioinformatics Toolbox™

User's Guide

R2020a
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Bioinformatics Toolbox™ User's Guide
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Revision History
September 2003 Online only New for Version 1.0 (Release 13SP1+)
June 2004 Online only Revised for Version 1.1 (Release 14)
November 2004 Online only Revised for Version 2.0 (Release 14SP1+)
March 2005 Online only Revised for Version 2.0.1 (Release 14SP2)
May 2005 Online only Revised for Version 2.1 (Release 14SP2+)
September 2005 Online only Revised for Version 2.1.1 (Release 14SP3)
November 2005 Online only Revised for Version 2.2 (Release 14SP3+)
March 2006 Online only Revised for Version 2.2.1 (Release 2006a)
May 2006 Online only Revised for Version 2.3 (Release 2006a+)
September 2006 Online only Revised for Version 2.4 (Release 2006b)
March 2007 Online only Revised for Version 2.5 (Release 2007a)
April 2007 Online only Revised for Version 2.6 (Release 2007a+)
September 2007 Online only Revised for Version 3.0 (Release 2007b)
March 2008 Online only Revised for Version 3.1 (Release 2008a)
October 2008 Online only Revised for Version 3.2 (Release 2008b)
March 2009 Online only Revised for Version 3.3 (Release 2009a)
September 2009 Online only Revised for Version 3.4 (Release 2009b)
March 2010 Online only Revised for Version 3.5 (Release 2010a)
September 2010 Online only Revised for Version 3.6 (Release 2010b)
April 2011 Online only Revised for Version 3.7 (Release 2011a)
September 2011 Online only Revised for Version 4.0 (Release 2011b)
March 2012 Online only Revised for Version 4.1 (Release 2012a)
September 2012 Online only Revised for Version 4.2 (Release 2012b)
March 2013 Online only Revised for Version 4.3 (Release 2013a)
September 2013 Online only Revised for Version 4.3.1 (Release 2013b)
March 2014 Online only Revised for Version 4.4 (Release 2014a)
October 2014 Online only Revised for Version 4.5 (Release 2014b)
March 2015 Online only Revised for Version 4.5.1 (Release 2015a)
September 2015 Online only Revised for Version 4.5.2 (Release 2015b)
March 2016 Online only Revised for Version 4.6 (Release 2016a)
September 2016 Online only Updated for Version 4.7 (Release 2016b)
March 2017 Online only Updated for Version 4.8 (Release 2017a)
September 2017 Online only Updated for Version 4.9 (Release 2017b)
March 2018 Online only Updated for Version 4.10 (Release 2018a)
September 2018 Online only Updated for Version 4.11 (Release 2018b)
March 2019 Online only Updated for Version 4.12 (Release 2019a)
September 2019 Online only Updated for Version 4.13 (Release 2019b)
March 2020 Online only Updated for Version 4.14 (Release 2020a)
 Contents

Getting Started
1
Bioinformatics Toolbox Product Description . . . . . . . . . . . . . . . . . . . . . . . 1-2
Key Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Product Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Expected Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Data Formats and Databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

Sequence Alignments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7

Sequence Utilities and Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8

Protein Property Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10

Microarray Data Analysis Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11

Microarray Data Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12

Mass Spectrometry Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13

Graph Theory Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15

Graph Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-16

Statistical Learning and Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17

Prototyping and Development Environment . . . . . . . . . . . . . . . . . . . . . . . 1-18

Data Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-19

Exchange Bioinformatics Data Between Excel and MATLAB . . . . . . . . . . 1-20

Using Excel and MATLAB Together . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-20
About the Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-20
Before Running the Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-20
Running the Example for the Entire Data Set . . . . . . . . . . . . . . . . . . . . . 1-21
Editing Formulas to Run the Example on a Subset of the Data . . . . . . . . 1-22
Using the Spreadsheet Link product to Interact With the Data in MATLAB
..................................................... 1-23

v
 Get Information from Web Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26
What Are get Functions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26
Creating the getpubmed Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26

High-Throughput Sequence Analysis

2
Work with Next-Generation Sequencing Data . . . . . . . . . . . . . . . . . . . . . . . 2-2
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
What Files Can You Access? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Create a BioIndexedFile Object to Access Your Source File . . . . . . . . . . . . 2-3
Determine the Number of Entries Indexed By a BioIndexedFile Object . . . 2-3
Retrieve Entries from Your Source File . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Read Entries from Your Source File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

Manage Sequence Read Data in Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Represent Sequence and Quality Data in a BioRead Object . . . . . . . . . . . . 2-7
Represent Sequence, Quality, and Alignment/Mapping Data in a BioMap
Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8
Retrieve Information from a BioRead or BioMap Object . . . . . . . . . . . . . 2-10
Set Information in a BioRead or BioMap Object . . . . . . . . . . . . . . . . . . . 2-12
Determine Coverage of a Reference Sequence . . . . . . . . . . . . . . . . . . . . 2-12
Construct Sequence Alignments to a Reference Sequence . . . . . . . . . . . 2-13
Filter Read Sequences Using SAM Flags . . . . . . . . . . . . . . . . . . . . . . . . . 2-14

Store and Manage Feature Annotations in Objects . . . . . . . . . . . . . . . . . 2-16

Represent Feature Annotations in a GFFAnnotation or GTFAnnotation
Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16
Construct an Annotation Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16
Retrieve General Information from an Annotation Object . . . . . . . . . . . . 2-16
Access Data in an Annotation Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-17
Use Feature Annotations with Sequence Read Data . . . . . . . . . . . . . . . . 2-18

Visualize and Investigate Sequence Read Alignments . . . . . . . . . . . . . . . 2-21

When to Use the NGS Browser to Visualize and Investigate Data . . . . . . 2-21
Open the NGS Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21
Import Data into the NGS Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23
Zoom and Pan to a Specific Region of the Alignment . . . . . . . . . . . . . . . . 2-25
View Coverage of the Reference Sequence . . . . . . . . . . . . . . . . . . . . . . . 2-25
View the Pileup View of Short Reads . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26
Compare Alignments of Multiple Data Sets . . . . . . . . . . . . . . . . . . . . . . . 2-26
View Location, Quality Scores, and Mapping Information . . . . . . . . . . . . 2-27
Flag Reads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28
Evaluate and Flag Mismatches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28
View Insertions and Deletions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29
View Feature Annotations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29
Print and Export the Browser Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-30

Count Features from NGS Reads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-31

vi Contents
 Identifying Differentially Expressed Genes from RNA-Seq Data . . . . . . . 2-41

Visualize NGS Data Using Genomics Viewer App . . . . . . . . . . . . . . . . . . . 2-69

Open the App . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-69
Add Tracks by Importing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-69
Visualize Single Nucleotide Variation in Cytochrome P450 . . . . . . . . . . . 2-70

Sequence Analysis
3
Exploring a Nucleotide Sequence Using Command Line . . . . . . . . . . . . . . 3-2
Overview of Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Searching the Web for Sequence Information . . . . . . . . . . . . . . . . . . . . . . 3-2
Reading Sequence Information from the Web . . . . . . . . . . . . . . . . . . . . . . 3-4
Determining Nucleotide Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
Determining Codon Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8
Open Reading Frames . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11
Amino Acid Conversion and Composition . . . . . . . . . . . . . . . . . . . . . . . . 3-13

Exploring a Nucleotide Sequence Using the Sequence Viewer App . . . . 3-15

Overview of the Sequence Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15
Importing a Sequence into the Sequence Viewer . . . . . . . . . . . . . . . . . . 3-15
Viewing Nucleotide Sequence Information . . . . . . . . . . . . . . . . . . . . . . . 3-17
Searching for Words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19
Exploring Open Reading Frames . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-22
Closing the Sequence Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-25

Explore a Protein Sequence Using the Sequence Viewer App . . . . . . . . . 3-26

Overview of the Sequence Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-26
Viewing Amino Acid Sequence Statistics . . . . . . . . . . . . . . . . . . . . . . . . . 3-26
Closing the Sequence Viewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-28
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-29

Compare Sequences Using Sequence Alignment Algorithms . . . . . . . . . 3-30

Overview of Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30
Find a Model Organism to Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30
Retrieve Sequence Information from a Public Database . . . . . . . . . . . . . 3-31
Search a Public Database for Related Genes . . . . . . . . . . . . . . . . . . . . . . 3-33
Locate Protein Coding Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-34
Compare Amino Acid Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-36

View and Align Multiple Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-43

Overview of the Sequence Alignment App . . . . . . . . . . . . . . . . . . . . . . . . 3-43
Visualize Multiple Sequence Alignment . . . . . . . . . . . . . . . . . . . . . . . . . . 3-43
Adjust Sequence Alignments Manually . . . . . . . . . . . . . . . . . . . . . . . . . . 3-44
Rearrange Rows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-52
Generate Phylogenetic Tree from Aligned Sequences . . . . . . . . . . . . . . . 3-54

vii
 Microarray Analysis
4
Managing Gene Expression Data in Objects . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Representing Expression Data Values in DataMatrix Objects . . . . . . . . . . 4-5

Overview of DataMatrix Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
Constructing DataMatrix Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
Getting and Setting Properties of a DataMatrix Object . . . . . . . . . . . . . . . 4-6
Accessing Data in DataMatrix Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Representing Expression Data Values in ExptData Objects . . . . . . . . . . . . 4-9

Overview of ExptData Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9
Constructing ExptData Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9
Using Properties of an ExptData Object . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
Using Methods of an ExptData Object . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11

Representing Sample and Feature Metadata in MetaData Objects . . . . . 4-12

Overview of MetaData Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12
Constructing MetaData Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Using Properties of a MetaData Object . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15
Using Methods of a MetaData Object . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15

Representing Experiment Information in a MIAME Object . . . . . . . . . . . 4-16

Overview of MIAME Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16
Constructing MIAME Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16
Using Properties of a MIAME Object . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17
Using Methods of a MIAME Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-18

Representing All Data in an ExpressionSet Object . . . . . . . . . . . . . . . . . . 4-19

Overview of ExpressionSet Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-19
Constructing ExpressionSet Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-20
Using Properties of an ExpressionSet Object . . . . . . . . . . . . . . . . . . . . . 4-21
Using Methods of an ExpressionSet Object . . . . . . . . . . . . . . . . . . . . . . . 4-21

Visualizing Microarray Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23

Overview of the Mouse Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23
Exploring the Microarray Data Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23
Spatial Images of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-25
Statistics of the Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-33
Scatter Plots of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-34

Phylogenetic Analysis
5
Using the Phylogenetic Tree App . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
Overview of the Phylogenetic Tree App . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
Opening the Phylogenetic Tree App . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3
Tools Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11

viii Contents
Window Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-17
Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18

ix
 1

Getting Started

• “Bioinformatics Toolbox Product Description” on page 1-2

• “Product Overview” on page 1-3
• “Data Formats and Databases” on page 1-5
• “Sequence Alignments” on page 1-7
• “Sequence Utilities and Statistics” on page 1-8
• “Protein Property Analysis” on page 1-9
• “Phylogenetic Analysis” on page 1-10
• “Microarray Data Analysis Tools” on page 1-11
• “Microarray Data Storage” on page 1-12
• “Mass Spectrometry Data Analysis” on page 1-13
• “Graph Theory Functions” on page 1-15
• “Graph Visualization” on page 1-16
• “Statistical Learning and Visualization” on page 1-17
• “Prototyping and Development Environment” on page 1-18
• “Data Visualization” on page 1-19
• “Exchange Bioinformatics Data Between Excel and MATLAB” on page 1-20
• “Get Information from Web Database” on page 1-26
1 Getting Started

Bioinformatics Toolbox Product Description

Read, analyze, and visualize genomic and proteomic data

Bioinformatics Toolbox provides algorithms and apps for Next Generation Sequencing (NGS),
microarray analysis, mass spectrometry, and gene ontology. Using toolbox functions, you can read
genomic and proteomic data from standard file formats such as SAM, FASTA, CEL, and CDF, as well
as from online databases such as the NCBI Gene Expression Omnibus and GenBank®. You can explore
and visualize this data with sequence browsers, spatial heatmaps, and clustergrams. The toolbox also
provides statistical techniques for detecting peaks, imputing values for missing data, and selecting
features.

You can combine toolbox functions to support common bioinformatics workflows. You can use ChIP-
Seq data to identify transcription factors; analyze RNA-Seq data to identify differentially expressed
genes; identify copy number variants and SNPs in microarray data; and classify protein profiles using
mass spectrometry data.

Key Features
• Next Generation Sequencing analysis and browser
• Sequence analysis and visualization, including pairwise and multiple sequence alignment and
peak detection
• Microarray data analysis, including reading, filtering, normalizing, and visualization
• Mass spectrometry analysis, including preprocessing, classification, and marker identification
• Phylogenetic tree analysis
• Graph theory functions, including interaction maps, hierarchy plots, and pathways
• Data import from genomic, proteomic, and gene expression files, including SAM, FASTA, CEL, and
CDF, and from databases such as NCBI and GenBank

1-2
 Product Overview

Product Overview

Features
The Bioinformatics Toolbox product extends the MATLAB® environment to provide an integrated
software environment for genome and proteome analysis. Scientists and engineers can answer
questions, solve problems, prototype new algorithms, and build applications for drug discovery and
design, genetic engineering, and biological research. An introduction to these features will help you
to develop a conceptual model for working with the toolbox and your biological data.

The Bioinformatics Toolbox product includes many functions to help you with genome and proteome
analysis. Most functions are implemented in the MATLAB programming language, with the source
available for you to view. This open environment lets you explore and customize the existing toolbox
algorithms or develop your own.

You can use the basic bioinformatic functions provided with this toolbox to create more complex
algorithms and applications. These robust and well-tested functions are the functions that you would
otherwise have to create yourself.

Toolbox features and functions fall within these categories:

• Data formats and databases — Connect to Web-accessible databases containing genomic and
proteomic data. Read and convert between multiple data formats.
• High-throughput sequencing — Gene expression and transcription factor analysis of next-
generation sequencing data, including RNA-Seq and ChIP-Seq.
• Sequence analysis — Determine the statistical characteristics of a sequence, align two
sequences, and multiply align several sequences. Model patterns in biological sequences using
hidden Markov model (HMM) profiles.
• Phylogenetic analysis — Create and manipulate phylogenetic tree data.
• Microarray data analysis — Read, normalize, and visualize microarray data.
• Mass spectrometry data analysis — Analyze and enhance raw mass spectrometry data.
• Statistical learning — Classify and identify features in data sets with statistical learning tools.
• Programming interface — Use other bioinformatic software (BioPerl and BioJava) within the
MATLAB environment.

The field of bioinformatics is rapidly growing and will become increasingly important as biology
becomes a more analytical science. The toolbox provides an open environment that you can customize
for development and deployment of the analytical tools you will need.

• Prototype and develop algorithms — Prototype new ideas in an open and extensible
environment. Develop algorithms using efficient string processing and statistical functions, view
the source code for existing functions, and use the code as a template for customizing, improving,
or creating your own functions. See “Prototyping and Development Environment” on page 1-18.
• Visualize data — Visualize sequences and alignments, gene expression data, phylogenetic trees,
mass spectrometry data, protein structure, and relationships between data with interconnected
graphs. See “Data Visualization” on page 1-19.
• Share and deploy applications — Use an interactive GUI builder to develop a custom graphical
front end for your data analysis programs. Create standalone applications that run separately
from the MATLAB environment.

1-3
1 Getting Started

Expected Users
The Bioinformatics Toolbox product is intended for computational biologists and research scientists
who need to develop new algorithms or implement published ones, visualize results, and create
standalone applications.

• Industry/Professional — Increasingly, drug discovery methods are being supported by

engineering practice. This toolbox supports tool builders who want to create applications for the
biotechnology and pharmaceutical industries.
• Education/Professor/Student — This toolbox is well suited for learning and teaching genome
and proteome analysis techniques. Educators and students can concentrate on bioinformatic
algorithms instead of programming basic functions such as reading and writing to files.

While the toolbox includes many bioinformatic functions, it is not intended to be a complete set of
tools for scientists to analyze their biological data. However, the MATLAB environment is ideal for
rapidly designing and prototyping the tools you need.

1-4
 Data Formats and Databases

Data Formats and Databases

The Bioinformatics Toolbox lets you access many of the databases on the web and other online data
repositories. It lets you copy data into the MATLAB workspace, and read and write to files with
standard bioinformatic formats. It also reads many common genome file formats so that you do not
have to write and maintain your own file readers.

Web-based databases — You can directly access public databases on the Web and copy sequence
and gene expression information into the MATLAB environment.

The sequence databases currently supported are GenBank (getgenbank), GenPept (getgenpept),
European Molecular Biology Laboratory (EMBL) (getembl), and Protein Data Bank (PDB) (getpdb).
You can also access data from the NCBI Gene Expression Omnibus (GEO) Web site by using a single
function (getgeodata).

Get multiply aligned sequences (gethmmalignment), hidden Markov model profiles (gethmmprof),
and phylogenetic tree data (gethmmtree) from the PFAM database.

Gene Ontology database — Load the database from the Web into a gene ontology object (geneont).
Select sections of the ontology with methods for the geneont object (getancestors (geneont),
getdescendants (geneont), getmatrix (geneont), getrelatives (geneont)), and
manipulate data with utility functions (goannotread, num2goid).

Read data from instruments — Read data generated from gene sequencing instruments (scfread,
joinseq, traceplot), mass spectrometers (jcampread), and Agilent® microarray scanners
(agferead).

Reading data formats — The toolbox provides a number of functions for reading data from common
bioinformatic file formats.

• Sequence data: GenBank (genbankread), GenPept (genpeptread), EMBL (emblread), PDB

(pdbread), and FASTA (fastaread)
• Multiply aligned sequences: ClustalW and GCG formats (multialignread)
• Gene expression data from microarrays: Gene Expression Omnibus (GEO) data (geosoftread),
GenePix® data in GPR and GAL files (gprread, galread), SPOT data (sptread), Affymetrix®
GeneChip® data (affyread), and ImaGene® results files (imageneread)
• Hidden Markov model profiles: PFAM-HMM file (pfamhmmread)

Writing data formats — The functions for getting data from the Web include the option to save the
data to a file. However, there is a function to write data to a file using the FASTA format
(fastawrite).

BLAST searches — Request Web-based BLAST searches (blastncbi), get the results from a search
(getblast) and read results from a previously saved BLAST formatted report file (blastread).

The MATLAB environment has built-in support for other industry-standard file formats including
Microsoft® Excel® and comma-separated-value (CSV) files. Additional functions perform ASCII and
low-level binary I/O, allowing you to develop custom functions for working with any data format.

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1 Getting Started

See Also

More About
• “High-Throughput Sequencing”
• “Microarray Analysis”
• “Sequence Analysis”
• “Structural Analysis”
• “Mass Spectrometry and Bioanalytics”

1-6
 Sequence Alignments

Sequence Alignments
You can select from a list of analysis methods to compare nucleotide or amino acid sequences using
pairwise or multiple sequence alignment functions.

Pairwise sequence alignment — Efficient implementations of standard algorithms such as the

Needleman-Wunsch (nwalign) and Smith-Waterman (swalign) algorithms for pairwise sequence
alignment. The toolbox also includes standard scoring matrices such as the PAM and BLOSUM
families of matrices (blosum, dayhoff, gonnet, nuc44, pam). Visualize sequence similarities with
seqdotplot and sequence alignment results with showalignment.

Multiple sequence alignment — Functions for multiple sequence alignment (multialign,

profalign) and functions that support multiple sequences (multialignread, fastaread,
showalignment). There is also a graphical interface (seqalignviewer) for viewing the results of a
multiple sequence alignment and manually making adjustment.

Multiple sequence profiles — Implementations for multiple alignment and profile hidden Markov
model algorithms (gethmmprof, gethmmalignment, gethmmtree, pfamhmmread, hmmprofalign,
hmmprofestimate, hmmprofgenerate, hmmprofmerge, hmmprofstruct, showhmmprof).

Biological codes — Look up the letters or numeric equivalents for commonly used biological codes
(aminolookup, baselookup, geneticcode, revgeneticcode).

See Also

More About
• “Sequence Utilities and Statistics” on page 1-8
• “Sequence Analysis”
• “Data Formats and Databases” on page 1-5

1-7
1 Getting Started

Sequence Utilities and Statistics

You can manipulate and analyze your sequences to gain a deeper understanding of the physical,
chemical, and biological characteristics of your data. Use a graphical user interface (GUI) with many
of the sequence functions in the toolbox (seqviewer).

Sequence conversion and manipulation — The toolbox provides routines for common operations,
such as converting DNA or RNA sequences to amino acid sequences, that are basic to working with
nucleic acid and protein sequences (aa2int, aa2nt, dna2rna, rna2dna, int2aa, int2nt, nt2aa,
nt2int, seqcomplement, seqrcomplement, seqreverse).

You can manipulate your sequence by performing an in silico digestion with restriction endonucleases
(restrict) and proteases (cleave).

Sequence statistics — Determine various statistics about a sequence (aacount, basecount,

codoncount, dimercount, nmercount, ntdensity, codonbias, cpgisland, oligoprop), search
for specific patterns within a sequence (seqshowwords, seqwordcount), or search for open reading
frames (seqshoworfs). In addition, you can create random sequences for test cases (randseq).

Sequence utilities — Determine a consensus sequence from a set of multiply aligned amino acid,
nucleotide sequences (seqconsensus, or a sequence profile (seqprofile). Format a sequence for
display (seqdisp) or graphically show a sequence alignment with frequency data (seqlogo).

Additional MATLAB functions efficiently handle string operations with regular expressions (regexp,
seq2regexp) to look for specific patterns in a sequence and search through a library for string
matches (seqmatch).

Look for possible cleavage sites in a DNA/RNA sequence by searching for palindromes
(palindromes).

See Also

More About
• “Sequence Alignments” on page 1-7
• “Sequence Analysis”
• “Protein and Amino Acid Sequence Analysis”
• “Data Formats and Databases” on page 1-5

1-8
 Protein Property Analysis

Protein Property Analysis

You can use a collection of protein analysis methods to extract information from your data. You can
determine protein characteristics and simulate enzyme cleavage reactions. The toolbox provides
functions to calculate various properties of a protein sequence, such as the atomic composition
(atomiccomp), molecular weight (molweight), and isoelectric point (isoelectric). You can cleave
a protein with an enzyme (cleave, rebasecuts) and create distance and Ramachandran plots for
PDB data (pdbdistplot, ramachandran). The toolbox contains a graphical user interface for
protein analysis (proteinplot) and plotting 3-D protein and other molecular structures with
information from molecule model files, such as PDB files (molviewer).

Amino acid sequence utilities — Calculate amino acid statistics for a sequence (aacount) and get
information about character codes (aminolookup).

See Also

More About
• “Protein and Amino Acid Sequence Analysis”
• “Structural Analysis”

1-9
1 Getting Started

Phylogenetic Analysis
Phylogenetic analysis is the process you use to determine the evolutionary relationships between
organisms. The results of an analysis can be drawn in a hierarchical diagram called a cladogram or
phylogram (phylogenetic tree). The branches in a tree are based on the hypothesized evolutionary
relationships (phylogeny) between organisms. Each member in a branch, also known as a
monophyletic group, is assumed to be descended from a common ancestor. Originally, phylogenetic
trees were created using morphology, but now, determining evolutionary relationships includes
matching patterns in nucleic acid and protein sequences. The Bioinformatics Toolbox provides the
following data structure and functions for phylogenetic analysis.

Phylogenetic tree data — Read and write Newick-formatted tree files (phytreeread,
phytreewrite) into the MATLAB Workspace as phylogenetic tree objects (phytree).

Create a phylogenetic tree — Calculate the pairwise distance between biological sequences
(seqpdist), estimate the substitution rates (dnds, dndsml), build a phylogenetic tree from pairwise
distances (seqlinkage, seqneighjoin, reroot), and view the tree in an interactive GUI that
allows you to view, edit, and explore the data (phytreeviewer or view). This GUI also allows you to
prune branches, reorder, rename, and explore distances.

Phylogenetic tree object methods — You can access the functionality of the phytreeviewer user
interface using methods for a phylogenetic tree object (phytree). Get property values (get) and
node names (getbyname). Calculate the patristic distances between pairs of leaf nodes (pdist,
weights) and draw a phylogenetic tree object in a MATLAB Figure window as a phylogram,
cladogram, or radial treeplot (plot). Manipulate tree data by selecting branches and leaves using a
specified criterion (select, subtree) and removing nodes (prune). Compare trees (getcanonical)
and use Newick-formatted strings (getnewickstr).

See Also

More About
• “Sequence Utilities and Statistics” on page 1-8
• “Sequence Analysis”

1-10
 Microarray Data Analysis Tools

Microarray Data Analysis Tools

The MATLAB environment is widely used for microarray data analysis, including reading, filtering,
normalizing, and visualizing microarray data. However, the standard normalization and visualization
tools that scientists use can be difficult to implement. The toolbox includes these standard functions:

Microarray data — Read Affymetrix GeneChip files (affyread) and plot data (probesetplot),
ImaGene results files (imageneread), SPOT files (sptread) and Agilent microarray scanner files
(agferead). Read GenePix GPR files (gprread) and GAL files (galread). Get Gene Expression
Omnibus (GEO) data from the Web (getgeodata) and read GEO data from files (geosoftread).

A utility function (magetfield) extracts data from one of the microarray reader functions (gprread,
agferead, sptread, imageneread).

Microarray normalization and filtering — The toolbox provides a number of methods for
normalizing microarray data, such as lowess normalization (malowess) and mean normalization
(manorm), or across multiple arrays (quantilenorm). You can use filtering functions to clean raw
data before analysis (geneentropyfilter, genelowvalfilter, generangefilter,
genevarfilter), and calculate the range and variance of values (exprprofrange, exprprofvar).

Microarray visualization — The toolbox contains routines for visualizing microarray data. These
routines include spatial plots of microarray data (maimage, redgreencmap), box plots (maboxplot),
loglog plots (maloglog), and intensity-ratio plots (mairplot). You can also view clustered expression
profiles (clustergram, redgreencmap). You can create 2-D scatter plots of principal components
from the microarray data (mapcaplot).

Microarray utility functions — Use the following functions to work with Affymetrix GeneChip data
sets. Get library information for a probe (probelibraryinfo), gene information from a probe set
(probesetlookup), and probe set values from CEL and CDF information (probesetvalues). Show
probe set information from NetAffx™ Analysis Center (probesetlink) and plot probe set values
(probesetplot).

The toolbox accesses statistical routines to perform cluster analysis and to visualize the results, and
you can view your data through statistical visualizations such as dendrograms, classification, and
regression trees.

See Also

More About
• “Microarray Data Storage” on page 1-12
• “Microarray Analysis”

1-11
1 Getting Started

Microarray Data Storage

The Bioinformatics Toolbox includes functions, objects, and methods for creating, storing, and
accessing microarray data.

The object constructor function, DataMatrix, lets you create a DataMatrix object to encapsulate
data and metadata from a microarray experiment. A DataMatrix object stores experimental data in a
matrix, with rows typically corresponding to gene names or probe identifiers, and columns typically
corresponding to sample identifiers. A DataMatrix object also stores metadata, including the gene
names or probe identifiers (as the row names) and sample identifiers (as the column names).

You can reference microarray expression values in a DataMatrix object the same way you reference
data in a MATLAB array, that is, by using linear or logical indexing. Alternately, you can reference this
experimental data by gene (probe) identifiers and sample identifiers. Indexing by these identifiers lets
you quickly and conveniently access subsets of the data without having to maintain additional index
arrays.

Many MATLAB operators and arithmetic functions are available to DataMatrix objects by means of
methods. These methods let you modify, combine, compare, analyze, plot, and access information
from DataMatrix objects. Additionally, you can easily extend the functionality by using general
element-wise functions, dmarrayfun and dmbsxfun, and by manually accessing the properties of a
DataMatrix object.

Note For more information on creating and using DataMatrix objects, see “Representing Expression
Data Values in DataMatrix Objects” on page 4-5.

See Also

More About
• “Microarray Data Analysis Tools” on page 1-11
• “Microarray Analysis”

1-12
 Mass Spectrometry Data Analysis

Mass Spectrometry Data Analysis

The mass spectrometry functions preprocess and classify raw data from SELDI-TOF and MALDI-TOF
spectrometers and use statistical learning functions to identify patterns.

Reading raw data — Load raw mass/charge and ion intensity data from comma-separated-value
(CSV) files, or read a JCAMP-DX-formatted file with mass spectrometry data (jcampread) into the
MATLAB environment.

You can also have data in TXT files and use the importdata function.

Preprocessing raw data — Resample high-resolution data to a lower resolution (msresample)

where the extra data points are not needed. Correct the baseline (msbackadj). Align a spectrum to a
set of reference masses (msalign) and visually verify the alignment (msheatmap). Normalize the
area between spectra for comparing (msnorm), and filter out noise (mslowess and mssgolay).

Spectrum analysis — Load spectra into a GUI (msviewer) for selecting mass peaks and further
analysis.

The following graphic illustrates the roles of the various mass spectrometry functions in the toolbox.

1-13
1 Getting Started

See Also

More About
• “Mass Spectrometry and Bioanalytics”
• “Data Formats and Databases” on page 1-5

1-14
 Graph Theory Functions

Graph Theory Functions

Graph theory functions in the Bioinformatics Toolbox apply basic graph theory algorithms to sparse
matrices. A sparse matrix represents a graph, any nonzero entries in the matrix represent the edges
of the graph, and the values of these entries represent the associated weight (cost, distance, length,
or capacity) of the edge. Graph algorithms that use the weight information will cancel the edge if a
NaN or an Inf is found. Graph algorithms that do not use the weight information will consider the
edge if a NaN or an Inf is found, because these algorithms look only at the connectivity described by
the sparse matrix and not at the values stored in the sparse matrix.

Sparse matrices can represent four types of graphs:

• Directed Graph — Sparse matrix, either double real or logical. Row (column) index indicates the
source (target) of the edge. Self-loops (values in the diagonal) are allowed, although most of the
algorithms ignore these values.
• Undirected Graph — Lower triangle of a sparse matrix, either double real or logical. An
algorithm expecting an undirected graph ignores values stored in the upper triangle of the sparse
matrix and values in the diagonal.
• Direct Acyclic Graph (DAG) — Sparse matrix, double real or logical, with zero values in the
diagonal. While a zero-valued diagonal is a requirement of a DAG, it does not guarantee a DAG. An
algorithm expecting a DAG will not test for cycles because this will add unwanted complexity.
• Spanning Tree — Undirected graph with no cycles and with one connected component.

There are no attributes attached to the graphs; sparse matrices representing all four types of graphs
can be passed to any graph algorithm. All functions will return an error on nonsquare sparse
matrices.

Graph algorithms do not pretest for graph properties because such tests can introduce a time penalty.
For example, there is an efficient shortest path algorithm for DAG, however testing if a graph is
acyclic is expensive compared to the algorithm. Therefore, it is important to select a graph theory
function and properties appropriate for the type of the graph represented by your input matrix. If the
algorithm receives a graph type that differs from what it expects, it will either:

• Return an error when it reaches an inconsistency. For example, if you pass a cyclic graph to the
graphshortestpath function and specify Acyclic as the method property.
• Produce an invalid result. For example, if you pass a directed graph to a function with an
algorithm that expects an undirected graph, it will ignore values in the upper triangle of the
sparse matrix.

The graph theory functions include graphallshortestpaths, graphconncomp, graphisdag,

graphisomorphism, graphisspantree, graphmaxflow, graphminspantree, graphpred2path,
graphshortestpath, graphtopoorder, and graphtraverse.

See Also

More About
• “Graph Visualization” on page 1-16
• “Network Analysis and Visualization”

1-15
1 Getting Started

Graph Visualization
The Bioinformatics Toolbox includes functions, objects, and methods for creating, viewing, and
manipulating graphs such as interactive maps, hierarchy plots, and pathways. This allows you to view
relationships between data.

The object constructor function (biograph) lets you create a biograph object to hold graph data.
Methods of the biograph object let you calculate the position of nodes (dolayout), draw the graph
(view), get handles to the nodes and edges (getnodesbyid and getedgesbynodeid) to further
query information, and find relations between the nodes (getancestors, getdescendants, and
getrelatives). There are also methods that apply basic graph theory algorithms to the biograph
object.

Various properties of a biograph object let you programmatically change the properties of the
rendered graph. You can customize the node representation, for example, drawing pie charts inside
every node (CustomNodeDrawFcn). Or you can associate your own callback functions to nodes and
edges of the graph, for example, opening a Web page with more information about the nodes
(NodeCallback and EdgeCallback).

See Also

More About
• “Graph Theory Functions” on page 1-15
• “Network Analysis and Visualization”

1-16
 Statistical Learning and Visualization

Statistical Learning and Visualization

You can classify and identify features in data sets, set up cross-validation experiments, and compare
different classification methods.

The toolbox provides functions that build on the classification and statistical learning tools in the
Statistics and Machine Learning Toolbox™ software (classify, kmeans, fitctree, and
fitrtree).

These functions include imputation tools (knnimpute), and K-nearest neighbor classifiers (fitcknn).

Other functions include set up of cross-validation experiments (crossvalind) and comparison of the
performance of different classification methods (classperf). In addition, there are tools for
selecting diversity and discriminating features (rankfeatures, randfeatures).

1-17
1 Getting Started

Prototyping and Development Environment

The MATLAB environment lets you prototype and develop algorithms and easily compare alternatives.

• Integrated environment — Explore biological data in an environment that integrates

programming and visualization. Create reports and plots with the built-in functions for
mathematics, graphics, and statistics.
• Open environment — Access the source code for the toolbox functions. The toolbox includes
many of the basic bioinformatics functions you will need to use, and it includes prototypes for
some of the more advanced functions. Modify these functions to create your own custom solutions.
• Interactive programming language — Test your ideas by typing functions that are interpreted
interactively with a language whose basic data element is an array. The arrays do not require
dimensioning and allow you to solve many technical computing problems,

Using matrices for sequences or groups of sequences allows you to work efficiently and not worry
about writing loops or other programming controls.
• Programming tools — Use a visual debugger for algorithm development and refinement and an
algorithm performance profiler to accelerate development.

1-18
 Data Visualization

Data Visualization
You can visually compare pairwise sequence alignments, multiply aligned sequences, gene expression
data from microarrays, and plot nucleic acid and protein characteristics. The 2-D and volume
visualization features let you create custom graphical representations of multidimensional data sets.
You can also create montages and overlays, and export finished graphics to an Adobe® PostScript®
image file or copy directly into Microsoft PowerPoint®.

1-19
1 Getting Started

Exchange Bioinformatics Data Between Excel and MATLAB

In this section...
“Using Excel and MATLAB Together” on page 1-20
“About the Example” on page 1-20
“Before Running the Example” on page 1-20
“Running the Example for the Entire Data Set” on page 1-21
“Editing Formulas to Run the Example on a Subset of the Data” on page 1-22
“Using the Spreadsheet Link product to Interact With the Data in MATLAB” on page 1-23

Using Excel and MATLAB Together

If you have bioinformatics data in an Excel (2007 or newer) spreadsheet, use Spreadsheet Link to:

• Connect Excel with the MATLAB Workspace to exchange data

• Use MATLAB and Bioinformatics Toolbox computational and visualization functions

About the Example

Note The following example assumes you have Spreadsheet Link software installed on your system.

The Excel file used in the following example contains data from DeRisi, J.L., Iyer, V.R., and Brown, P.O.
(Oct. 24, 1997). Exploring the metabolic and genetic control of gene expression on a genomic scale.
Science 278(5338), 680–686. PMID: 9381177. The data was filtered using the steps described in
“Gene Expression Profile Analysis”.

Before Running the Example

1 If not already done, modify your system path to include the MATLAB root folder as described in
the Spreadsheet Link documentation.
2 If not already done, enable the Spreadsheet Link Add-In as described in “Add-In Setup”
(Spreadsheet Link).
3 Close MATLAB and Excel if they are open.
4 Start Excel. MATLAB and Spreadsheet Link software automatically start.
5 From Excel, open the following file provided with the Bioinformatics Toolbox software:

matlabroot\toolbox\bioinfo\biodemos\Filtered_Yeastdata.xlsm

Note matlabroot is the MATLAB root folder, which is where MATLAB software is installed on
your system.
6 In the Excel software, enable macros. Click the Developer tab, and then select Macro Security
from the Code group. If the Developer tab is not displayed on the Excel ribbon, consult Excel
Help to display it. If you encounter the "Can't find project or library" error, you might need to
update the references in the Visual Basic software. Open Visual Basic by clicking the Developer

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 Exchange Bioinformatics Data Between Excel and MATLAB

tab and selecting Visual Basic. Then select Tools > References > SpreadsheetLink. If the
MISSING: exclink2007.xlam check box is selected, clear it.

Running the Example for the Entire Data Set

1 In the provided Excel file, note that columns A through H contain data from DeRisi et al. Also
note that cells J5, J6, J7, and J12 contain formulas using Spreadsheet Link functions
MLPutMatrix and MLEvalString.

Tip To view a cell's formula, select the cell, and then view the formula in the formula bar
at the top of the Excel window.
2 Execute the formulas in cells J5, J6, J7, and J12, by selecting the cell, pressing F2, and then
pressing Enter.

Each of the first three cells contains a formula using the Spreadsheet Link function
MLPutMatrix, which creates a MATLAB variable from the data in the spreadsheet. Cell J12
contains a formula using the Spreadsheet Link function MLEvalString, which runs the
Bioinformatics Toolbox clustergram function using the three variables as input. For more
information on adding formulas using Spreadsheet Link functions, see “Create Diagonal Matrix
Using Worksheet Cells” (Spreadsheet Link).

1-21
1 Getting Started

3 Note that cell J17 contains a formula using a macro function Clustergram, which was created in
the Visual Basic® Editor. Running this macro does the same as the formulas in cells J5, J6, J7, and
J12. Optionally, view the Clustergram macro function by clicking the Developer tab, and then

clicking the Visual Basic button . (If the Developer tab is not on the Excel ribbon, consult
Excel Help to display it.)

For more information on creating macros using Visual Basic Editor, see “Create Diagonal Matrix
Using VBA Macro” (Spreadsheet Link).
4 Execute the formula in cell J17 to analyze and visualize the data:

a Select cell J17.

b Press F2.
c Press Enter.

The macro function Clustergram runs creating three MATLAB variables (data, Genes, and
TimeSteps) and displaying a Clustergram window containing dendrograms and a heat map of
the data.

Editing Formulas to Run the Example on a Subset of the Data

1 Edit the formulas in cells J5 and J6 to analyze a subset of the data. Do this by editing the
formulas’ cell ranges to include data for only the first 30 genes:

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 Exchange Bioinformatics Data Between Excel and MATLAB

a Select cell J5, and then press F2 to display the formula for editing. Change H617 to H33,
and then press Enter.

b Select cell J6, then press F2 to display the formula for editing. Change A617 to A33, and
then press Enter.

2 Run the formulas in cells J5, J6, J7, and J12 to analyze and visualize a subset of the data:

a Select cell J5, press F2, and then press Enter.

b Select cell J6, press F2, and then press Enter.
c Select cell J7, press F2, and then press Enter.
d Select cell J12, press F2, and then press Enter.

Using the Spreadsheet Link product to Interact With the Data in

MATLAB
Use the MATLAB group on the right side of the Home tab to interact with the data:

1-23
1 Getting Started

For example, create a variable in MATLAB containing a 3-by-7 matrix of the data, plot the data in a
Figure window, and then add the plot to your spreadsheet:

1 Click-drag to select cells B5 through H7.

2 From the MATLAB group, select Send data to MATLAB.

3 Type YAGenes for the variable name, and then click OK.

The variable YAGenes is added to the MATLAB Workspace as a 3-by-7 matrix.

4 From the MATLAB group, select Run MATLAB command.
5 Type plot(YAGenes') for the command, and then click OK.

A Figure window displays a plot of the data.

Note Make sure you use the ' (transpose) symbol when plotting the data in this step. You need
to transpose the data in YAGenes so that it plots as three genes over seven time intervals.
6 Select cell J20, and then click from the MATLAB group, select Get MATLAB figure.

The figure is added to the spreadsheet.

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Exchange Bioinformatics Data Between Excel and MATLAB

1-25
1 Getting Started

Get Information from Web Database

In this section...
“What Are get Functions?” on page 1-26
“Creating the getpubmed Function” on page 1-26

What Are get Functions?

Bioinformatics Toolbox includes several get functions that retrieve information from various Web
databases. Additionally, with some basic MATLAB programming skills, you can create your own get
function to retrieve information from a specific Web database.

The following procedure illustrates how to create a function to retrieve information from the NCBI
PubMed database and read the information into a MATLAB structure. The NCBI PubMed database
contains biomedical literature citations and abstracts.

Creating the getpubmed Function

The following procedure shows you how to create a function named getpubmed using the MATLAB
Editor. This function will retrieve citation and abstract information from PubMed literature searches
and write the data to a MATLAB structure.

Specifically, this function will take one or more search terms, submit them to the PubMed database
for a search, then return a MATLAB structure or structure array, with each structure containing
information for an article found by the search. The returned information will include a PubMed
identifier, publication date, title, abstract, authors, and citation.

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 Get Information from Web Database

The function will also include property name-value pairs that let the user of the function limit the
search by publication date and limit the number of records returned. Below is the step-by-step guide
to create the function from the beginning. To see the completed m-file, type edit getpubmed.m.

1 From MATLAB, open the MATLAB Editor by selecting File > New > Function.
2 Define the getpubmed function, its input arguments, and return values by typing:

function pmstruct = getpubmed(searchterm,varargin)

% GETPUBMED Search PubMed database & write results to MATLAB structure
3 Add code to do some basic error checking for the required input SEARCHTERM.

% Error checking for required input SEARCHTERM

if(nargin<1)
error(message('bioinfo:getpubmed:NotEnoughInputArguments'));
end
4 Create variables for the two property name-value pairs, and set their default values.

% Set default settings for property name/value pairs,

% 'NUMBEROFRECORDS' and 'DATEOFPUBLICATION'
maxnum = 50; % NUMBEROFRECORDS default is 50
pubdate = ''; % DATEOFPUBLICATION default is an empty string
5 Add code to parse the two property name-value pairs if provided as input.

% Parsing the property name/value pairs

num_argin = numel(varargin);
for n = 1:2:num_argin
arg = varargin{n};
switch lower(arg)

% If NUMBEROFRECORDS is passed, set MAXNUM

case 'numberofrecords'
maxnum = varargin{n+1};

% If DATEOFPUBLICATION is passed, set PUBDATE

case 'dateofpublication'
pubdate = varargin{n+1};

end
end
6 You access the PubMed database through a search URL, which submits a search term and
options, and then returns the search results in a specified format. This search URL is comprised
of a base URL and defined parameters. Create a variable containing the base URL of the PubMed
database on the NCBI Web site.

% Create base URL for PubMed db site

baseSearchURL = 'https://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search';
7 Create variables to contain five defined parameters that the getpubmed function will use,
namely, db (database), term (search term), report (report type, such as MEDLINE®), format
(format type, such as text), and dispmax (maximum number of records to display).

% Set db parameter to pubmed

dbOpt = '&db=pubmed';

% Set term parameter to SEARCHTERM and PUBDATE

% (Default PUBDATE is '')

1-27
1 Getting Started

termOpt = ['&term=',searchterm,'+AND+',pubdate];

% Set report parameter to medline

reportOpt = '&report=medline';

% Set format parameter to text

formatOpt = '&format=text';

% Set dispmax to MAXNUM

% (Default MAXNUM is 50)
maxOpt = ['&dispmax=',num2str(maxnum)];
8 Create a variable containing the search URL from the variables created in the previous steps.

% Create search URL

searchURL = [baseSearchURL,dbOpt,termOpt,reportOpt,formatOpt,maxOpt];
9 Use the urlread function to submit the search URL, retrieve the search results, and return the
results (as text in the MEDLINE report type) in medlineText, a character array.

medlineText = urlread(searchURL);
10 Use the MATLAB regexp function and regular expressions to parse and extract the information
in medlineText into hits, a cell array, where each cell contains the MEDLINE-formatted text
for one article. The first input is the character array to search, the second input is a search
expression, which tells the regexp function to find all records that start with PMID-, while the
third input, 'match', tells the regexp function to return the actual records, rather than the
positions of the records.

hits = regexp(medlineText,'PMID-.*?(?=PMID|</pre>$)','match');
11 Instantiate the pmstruct structure returned by getpubmed to contain six fields.

pmstruct = struct('PubMedID','','PublicationDate','','Title','',...
'Abstract','','Authors','','Citation','');
12 Use the MATLAB regexp function and regular expressions to loop through each article in hits
and extract the PubMed ID, publication date, title, abstract, authors, and citation. Place this
information in the pmstruct structure array.
for n = 1:numel(hits)
pmstruct(n).PubMedID = regexp(hits{n},'(?<=PMID- ).*?(?=\n)','match', 'once');
pmstruct(n).PublicationDate = regexp(hits{n},'(?<=DP - ).*?(?=\n)','match', 'once');
pmstruct(n).Title = regexp(hits{n},'(?<=TI - ).*?(?=PG -|AB -)','match', 'once');
pmstruct(n).Abstract = regexp(hits{n},'(?<=AB - ).*?(?=AD -)','match', 'once');
pmstruct(n).Authors = regexp(hits{n},'(?<=AU - ).*?(?=\n)','match');
pmstruct(n).Citation = regexp(hits{n},'(?<=SO - ).*?(?=\n)','match', 'once');
end
13 Select File > Save As.

When you are done, your file should look similar to the getpubmed.m file included with the
Bioinformatics Toolbox software. The file is located at:

matlabroot\toolbox\bioinfo\biodemos\getpubmed.m

Note The notation matlabroot is the MATLAB root directory, which is the directory where the
MATLAB software is installed on your system.

1-28
 2

High-Throughput Sequence Analysis

• “Work with Next-Generation Sequencing Data” on page 2-2

• “Manage Sequence Read Data in Objects” on page 2-6
• “Store and Manage Feature Annotations in Objects” on page 2-16
• “Visualize and Investigate Sequence Read Alignments” on page 2-21
• “Count Features from NGS Reads” on page 2-31
• “Identifying Differentially Expressed Genes from RNA-Seq Data” on page 2-41
• “Visualize NGS Data Using Genomics Viewer App” on page 2-69
2 High-Throughput Sequence Analysis

Work with Next-Generation Sequencing Data

In this section...
“Overview” on page 2-2
“What Files Can You Access?” on page 2-2
“Before You Begin” on page 2-3
“Create a BioIndexedFile Object to Access Your Source File” on page 2-3
“Determine the Number of Entries Indexed By a BioIndexedFile Object” on page 2-3
“Retrieve Entries from Your Source File” on page 2-4
“Read Entries from Your Source File” on page 2-4

Overview
Many biological experiments produce huge data files that are difficult to access due to their size,
which can cause memory issues when reading the file into the MATLAB Workspace. You can construct
a BioIndexedFile object to access the contents of a large text file containing nonuniform size
entries, such as sequences, annotations, and cross-references to data sets. The BioIndexedFile
object lets you quickly and efficiently access this data without loading the source file into memory.

You can use the BioIndexedFile object to access individual entries or a subset of entries when the
source file is too big to fit into memory. You can access entries using indices or keys. You can read and
parse one or more entries using provided interpreters or a custom interpreter function.

Use the BioIndexedFile object in conjunction with your large source file to:

• Access a subset of the entries for validation or further analysis.

• Parse entries using a custom interpreter function.

What Files Can You Access?

You can use the BioIndexedFile object to access large text files.

Your source file can have these application-specific formats:

• FASTA
• FASTQ
• SAM

Your source file can also have these general formats:

• Table — Tab-delimited table with multiple columns. Keys can be in any column. Rows with the
same key are considered separate entries.
• Multi-row Table — Tab-delimited table with multiple columns. Keys can be in any column.
Contiguous rows with the same key are considered a single entry. Noncontiguous rows with the
same key are considered separate entries.
• Flat — Flat file with concatenated entries separated by a character vector, typically //. Within an
entry, the key is separated from the rest of the entry by a white space.

2-2
 Work with Next-Generation Sequencing Data

Before You Begin

Before constructing a BioIndexedFile object, locate your source file on your hard drive or a local
network.

When you construct a BioIndexedFile object from your source file for the first time, you also
create an auxiliary index file, which by default is saved to the same location as your source file.
However, if your source file is in a read-only location, you can specify a different location to save the
index file.

Tip If you construct a BioIndexedFile object from your source file on subsequent occasions, it
takes advantage of the existing index file, which saves time. However, the index file must be in the
same location or a location specified by the subsequent construction syntax.

Tip If insufficient memory is not an issue when accessing your source file, you may want to try an
appropriate read function, such as genbankread, for importing data from GenBank files. .

Additionally, several read functions such as fastaread, fastqread, samread, and sffread include
a Blockread property, which lets you read a subset of entries from a file, thus saving memory.

Create a BioIndexedFile Object to Access Your Source File

To construct a BioIndexedFile object from a multi-row table file:
1 Create a variable containing the full absolute path of your source file. For your source file, use
the yeastgenes.sgd file, which is included with the Bioinformatics Toolbox software.
sourcefile = which('yeastgenes.sgd');
2 Use the BioIndexedFile constructor function to construct a BioIndexedFile object from the
yeastgenes.sgd source file, which is a multi-row table file. Save the index file in the Current
Folder. Indicate that the source file keys are in column 3. Also, indicate that the header lines in
the source file are prefaced with !, so the constructor ignores them.
gene2goObj = BioIndexedFile('mrtab', sourcefile, '.', ...
'KeyColumn', 3, 'HeaderPrefix','!')

The BioIndexedFile constructor function constructs gene2goObj, a BioIndexedFile object,

and also creates an index file with the same name as the source file, but with an IDX extension. It
stores this index file in the Current Folder because we specified this location. However, the
default location for the index file is the same location as the source file.

Caution Do not modify the index file. If you modify it, you can get invalid results. Also, the
constructor function cannot use a modified index file to construct future objects from the
associated source file.

Determine the Number of Entries Indexed By a BioIndexedFile Object

To determine the number of entries indexed by a BioIndexedFile object, use the NumEntries
property of the BioIndexedFile object. For example, for the gene2goObj object:
gene2goObj.NumEntries

2-3
2 High-Throughput Sequence Analysis

ans =

6476

Note For a list and description of all properties of a BioIndexedFile object, see BioIndexedFile
class.

Retrieve Entries from Your Source File

Retrieve entries from your source file using either:

• The index of the entry

• The entry key

Retrieve Entries Using Indices

Use the getEntryByIndex method to retrieve a subset of entries from your source file that
correspond to specified indices. For example, retrieve the first 12 entries from the yeastgenes.sgd
source file:

subset_entries = getEntryByIndex(gene2goObj, [1:12]);

Retrieve Entries Using Keys

Use the getEntryByKey method to retrieve a subset of entries from your source file that are
associated with specified keys. For example, retrieve all entries with keys of AAC1 and AAD10 from
the yeastgenes.sgd source file:

subset_entries = getEntryByKey(gene2goObj, {'AAC1' 'AAD10'});

The output subset_entries is a character vector of concatenated entries. Because the keys in the
yeastgenes.sgd source file are not unique, this method returns all entries that have a key of AAC1
or AAD10.

Read Entries from Your Source File

The BioIndexedFile object includes a read method, which you can use to read and parse a subset
of entries from your source file. The read method parses the entries using an interpreter function
specified by the Interpreter property of the BioIndexedFile object.

Set the Interpreter Property

Before using the read method, make sure the Interpreter property of the BioIndexedFile
object is set appropriately.

If you constructed a BioIndexedFile The Interpreter property ...

object from ...
A source file with an application-specific By default is a handle to a function appropriate for that
format (FASTA, FASTQ, or SAM) file type and typically does not require you to change it.

2-4
 Work with Next-Generation Sequencing Data

If you constructed a BioIndexedFile The Interpreter property ...

object from ...
A source file with a table, multi-row table, or By default is [], which means the interpreter is an
flat format anonymous function in which the output is equivalent to
the input. You can change this to a handle to a function
that accepts a character vector of one or more
concatenated entries and returns a structure or an
array of structures containing the interpreted data.

There are two ways to set the Interpreter property of the BioIndexedFile object:

• When constructing the BioIndexedFile object, use the Interpreter property name/property
value pair
• After constructing the BioIndexedFile object, set the Interpreter property

Note For more information on setting the Interpreter property of a BioIndexedFile object, see
BioIndexedFile class.

Read a Subset of Entries

The read method reads and parses a subset of entries that you specify using either entry indices or
keys.

Example

To quickly find all the gene ontology (GO) terms associated with a particular gene because the entry
keys are gene names:

1 Set the Interpreter property of the gene2goObj BioIndexedFile object to a handle to a

function that reads entries and returns only the column containing the GO term. In this case the
interpreter is a handle to an anonymous function that accepts character vectors and extracts
those that start with the characters GO.

gene2goObj.Interpreter = @(x) regexp(x,'GO:\d+','match')

2 Read only the entries that have a key of YAT2, and return their GO terms.

GO_YAT2_entries = read(gene2goObj, 'YAT2')

GO_YAT2_entries =

'GO:0004092' 'GO:0005737' 'GO:0006066' 'GO:0006066' 'GO:0009437'

2-5
2 High-Throughput Sequence Analysis

Manage Sequence Read Data in Objects

In this section...
“Overview” on page 2-6
“Represent Sequence and Quality Data in a BioRead Object” on page 2-7
“Represent Sequence, Quality, and Alignment/Mapping Data in a BioMap Object” on page 2-8
“Retrieve Information from a BioRead or BioMap Object” on page 2-10
“Set Information in a BioRead or BioMap Object” on page 2-12
“Determine Coverage of a Reference Sequence” on page 2-12
“Construct Sequence Alignments to a Reference Sequence” on page 2-13
“Filter Read Sequences Using SAM Flags” on page 2-14

Overview
High-throughput sequencing instruments produce large amounts of sequence read data that can be
challenging to store and manage. Using objects to contain this data lets you easily access,
manipulate, and filter the data.

Bioinformatics Toolbox includes two objects for working with sequence read data.

Object Contains This Information Construct from One of These

BioRead • Sequence headers • FASTQ file
• Read sequences • SAM file
• Sequence qualities (base calling) • FASTQ structure (created using the
fastqread function)
• SAM structure (created using the
samread function)
• Cell arrays containing header,
sequence, and quality information
(created using the fastqread
function)
BioMap • Sequence headers • SAM file
• Read sequences • BAM file
• Sequence qualities (base calling) • SAM structure (created using the
• Sequence alignment and mapping samread function)
information (relative to a single • BAM structure (created using the
reference sequence), including bamread function)
mapping quality • Cell arrays containing header,
sequence, quality, and mapping/
alignment information (created using
the samread or bamread function)

2-6
 Manage Sequence Read Data in Objects

Represent Sequence and Quality Data in a BioRead Object

Prerequisites

A BioRead object represents a collection of sequence reads. Each element in the object is associated
with a sequence, sequence header, and sequence quality information.

Construct a BioRead object in one of two ways:

• Indexed — The data remains in the source file. Constructing the object and accessing its contents
is memory efficient. However, you cannot modify object properties, other than the Name property.
This is the default method if you construct a BioRead object from a FASTQ- or SAM-formatted
file.
• In Memory — The data is read into memory. Constructing the object and accessing its contents is
limited by the amount of available memory. However, you can modify object properties. When you
construct a BioRead object from a FASTQ structure or cell arrays, the data is read into memory.
When you construct a BioRead object from a FASTQ- or SAM-formatted file, use the InMemory
name-value pair argument to read the data into memory.

Construct a BioRead Object from a FASTQ- or SAM-Formatted File

Note This example constructs a BioRead object from a FASTQ-formatted file. Use similar steps to
construct a BioRead object from a SAM-formatted file.

Use the BioRead constructor function to construct a BioRead object from a FASTQ-formatted file
and set the Name property:

BRObj1 = BioRead('SRR005164_1_50.fastq', 'Name', 'MyObject')

BRObj1 =

BioRead with properties:

Quality: [50x1 File indexed property]

Sequence: [50x1 File indexed property]
Header: [50x1 File indexed property]
NSeqs: 50
Name: 'MyObject'

The constructor function construct a BioRead object and, if an index file does not already exist, it
also creates an index file with the same file name, but with an .IDX extension. This index file, by
default, is stored in the same location as the source file.

Caution Your source file and index file must always be in sync.

• After constructing a BioRead object, do not modify the index file, or you can get invalid results
when using the existing object or constructing new objects.
• If you modify the source file, delete the index file, so the object constructor creates a new index
file when constructing new objects.

2-7
2 High-Throughput Sequence Analysis

Note Because you constructed this BioRead object from a source file, you cannot modify the
properties (except for Name) of the BioRead object.

Represent Sequence, Quality, and Alignment/Mapping Data in a

BioMap Object
Prerequisites

A BioMap object represents a collection of sequence reads that map against a single reference
sequence. Each element in the object is associated with a read sequence, sequence header, sequence
quality information, and alignment/mapping information.

When constructing a BioMap object from a BAM file, the maximum size of the file is limited by your
operating system and available memory.

Construct a BioMap object in one of two ways:

• Indexed — The data remains in the source file. Constructing the object and accessing its contents
is memory efficient. However, you cannot modify object properties, other than the Name property.
This is the default method if you construct a BioMap object from a SAM- or BAM-formatted file.
• In Memory — The data is read into memory. Constructing the object and accessing its contents is
limited by the amount of available memory. However, you can modify object properties. When you
construct a BioMap object from a structure, the data stays in memory. When you construct a
BioMap object from a SAM- or BAM-formatted file, use the InMemory name-value pair argument
to read the data into memory.

Construct a BioMap Object from a SAM- or BAM-Formatted File

Note This example constructs a BioMap object from a SAM-formatted file. Use similar steps to
construct a BioMap object from a BAM-formatted file.

1 If you do not know the number and names of the reference sequences in your source file,
determine them using the saminfo or baminfo function and the ScanDictionary name-value
pair argument.

samstruct = saminfo('ex2.sam', 'ScanDictionary', true);

samstruct.ScannedDictionary

ans =

'seq1'
'seq2'

Tip The previous syntax scans the entire SAM file, which is time consuming. If you are confident
that the Header information of the SAM file is correct, omit the ScanDictionary name-value
pair argument, and inspect the SequenceDictionary field instead.
2 Use the BioMap constructor function to construct a BioMap object from the SAM file and set the
Name property. Because the SAM-formatted file in this example, ex2.sam, contains multiple
reference sequences, use the SelectRef name-value pair argument to specify one reference
sequence, seq1:

2-8
 Manage Sequence Read Data in Objects

BMObj2 = BioMap('ex2.sam', 'SelectRef', 'seq1', 'Name', 'MyObject')

BMObj2 =

BioMap with properties:

SequenceDictionary: 'seq1'
Reference: [1501x1 File indexed property]
Signature: [1501x1 File indexed property]
Start: [1501x1 File indexed property]
MappingQuality: [1501x1 File indexed property]
Flag: [1501x1 File indexed property]
MatePosition: [1501x1 File indexed property]
Quality: [1501x1 File indexed property]
Sequence: [1501x1 File indexed property]
Header: [1501x1 File indexed property]
NSeqs: 1501
Name: 'MyObject'

The constructor function constructs a BioMap object and, if index files do not already exist, it also
creates one or two index files:

• If constructing from a SAM-formatted file, it creates one index file that has the same file name as
the source file, but with an .IDX extension. This index file, by default, is stored in the same
location as the source file.
• If constructing from a BAM-formatted file, it creates two index files that have the same file name
as the source file, but one with a .BAI extension and one with a .LINEARINDEX extension. These
index files, by default, are stored in the same location as the source file.

Caution Your source file and index files must always be in sync.

• After constructing a BioMap object, do not modify the index files, or you can get invalid results
when using the existing object or constructing new objects.
• If you modify the source file, delete the index files, so the object constructor creates new index
files when constructing new objects.

Note Because you constructed this BioMap object from a source file, you cannot modify the
properties (except for Name and Reference) of the BioMap object.

Construct a BioMap Object from a SAM or BAM Structure

Note This example constructs a BioMap object from a SAM structure using samread. Use similar
steps to construct a BioMap object from a BAM structure using bamread.

1 Use the samread function to create a SAM structure from a SAM-formatted file:

SAMStruct = samread('ex2.sam');
2 To construct a valid BioMap object from a SAM-formatted file, the file must contain only one
reference sequence. Determine the number and names of the reference sequences in your SAM-

2-9
2 High-Throughput Sequence Analysis

formatted file using the unique function to find unique names in the ReferenceName field of
the structure:

unique({SAMStruct.ReferenceName})

ans =

'seq1' 'seq2'
3 Use the BioMap constructor function to construct a BioMap object from a SAM structure.
Because the SAM structure contains multiple reference sequences, use the SelectRef name-
value pair argument to specify one reference sequence, seq1:

BMObj1 = BioMap(SAMStruct, 'SelectRef', 'seq1')

BMObj1 =

BioMap with properties:

SequenceDictionary: {'seq1'}
Reference: {1501x1 cell}
Signature: {1501x1 cell}
Start: [1501x1 uint32]
MappingQuality: [1501x1 uint8]
Flag: [1501x1 uint16]
MatePosition: [1501x1 uint32]
Quality: {1501x1 cell}
Sequence: {1501x1 cell}
Header: {1501x1 cell}
NSeqs: 1501
Name: ''

Retrieve Information from a BioRead or BioMap Object

You can retrieve all or a subset of information from a BioRead or BioMap object.

Retrieve a Property from a BioRead or BioMap Object

You can retrieve a specific property from elements in a BioRead or BioMap object.

For example, to retrieve all headers from a BioRead object, use the Header property as follows:

allHeaders = BRObj1.Header;

This syntax returns a cell array containing the headers for all elements in the BioRead object.

Similarly, to retrieve all start positions of aligned read sequences from a BioMap object, use the
Start property of the object:

allStarts = BMObj1.Start;

This syntax returns a vector containing the start positions of aligned read sequences with respect to
the position numbers in the reference sequence in a BioMap object.

2-10
 Manage Sequence Read Data in Objects

Retrieve Multiple Properties from a BioRead or BioMap Object

You can retrieve multiple properties from a BioRead or BioMap object in a single command using the
get method. For example, to retrieve both start positions and headers information of a BioMap
object, use the get method as follows:

multiProp = get(BMObj1, {'Start', 'Header'});

This syntax returns a cell array containing all start positions and headers information of a BioMap
object.

Note Property names are case sensitive.

For a list and description of all properties of a BioRead object, see BioRead class. For a list and
description of all properties of a BioMap object, see BioMap class.

Retrieve a Subset of Information from a BioRead or BioMap Object

Use specialized get methods with a numeric vector, logical vector, or cell array of headers to retrieve
a subset of information from an object. For example, to retrieve the first 10 elements from a BioRead
object, use the getSubset method:

newBRObj = getSubset(BRObj1, [1:10]);

This syntax returns a new BioRead object containing the first 10 elements in the original BioRead
object.

For example, to retrieve the first 12 positions of sequences with headers SRR005164.1,
SRR005164.7, and SRR005164.16, use the getSubsequence method:

subSeqs = getSubsequence(BRObj1, ...

{'SRR005164.1', 'SRR005164.7', 'SRR005164.16'}, [1:12]')

subSeqs =

'TGGCTTTAAAGC'
'CCCGAAAGCTAG'
'AATTTTGCGGCT'

For example, to retrieve information about the third element in a BioMap object, use the getInfo
method:

Info_3 = getInfo(BMObj1, 3);

This syntax returns a tab-delimited character vector containing this information for the third element:

• Sequence header
• SAM flags for the sequence
• Start position of the aligned read sequence with respect to the reference sequence
• Mapping quality score for the sequence
• Signature (CIGAR-formatted character vector) for the sequence
• Sequence

2-11
2 High-Throughput Sequence Analysis

• Quality scores for sequence positions

Note Method names are case sensitive.

For a complete list and description of methods of a BioRead object, see BioRead class. For a
complete list and description of methods of a BioMap object, see BioMap class.

Set Information in a BioRead or BioMap Object

Prerequisites

To modify properties (other than Name and Reference) of a BioRead or BioMap object, the data
must be in memory, and not indexed. To ensure the data is in memory, do one of the following:

• Construct the object from a structure as described in “Construct a BioMap Object from a SAM or
BAM Structure” on page 2-9.
• Construct the object from a source file using the InMemory name-value pair argument.

Provide Custom Headers for Sequences

First, create an object with the data in memory:

BRObj1 = BioRead('SRR005164_1_50.fastq','InMemory',true);

To provide custom headers for sequences of interest (in this case sequences 1 to 5), do the following:

BRObj1.Header(1:5) = {'H1', 'H2', 'H3', 'H4', 'H5'};

Alternatively, you can use the setHeader method:

BRObj1 = setHeader(BRObj1, {'H1', 'H2', 'H3', 'H4', 'H5'}, [1:5]);

Several other specialized set methods let you set the properties of a subset of elements in a
BioRead or BioMap object.

Note Method names are case sensitive.

For a complete list and description of methods of a BioRead object, see BioRead class. For a
complete list and description of methods of a BioMap object, see BioMap class.

Determine Coverage of a Reference Sequence

When working with a BioMap object, you can determine the number of read sequences that:

• Align within a specific region of the reference sequence

• Align to each position within a specific region of the reference sequence

For example, you can compute the number, indices, and start positions of the read sequences that
align within the first 25 positions of the reference sequence. To do so, use the getCounts,
getIndex, and getStart methods:

Cov = getCounts(BMObj1, 1, 25)

2-12
 Manage Sequence Read Data in Objects

Cov =

12

Indices = getIndex(BMObj1, 1, 25)

Indices =

1
2
3
4
5
6
7
8
9
10
11
12

startPos = getStart(BMObj1, Indices)

startPos =

1
3
5
6
9
13
13
15
18
22
22
24

The first two syntaxes return the number and indices of the read sequences that align within the
specified region of the reference sequence. The last syntax returns a vector containing the start
position of each aligned read sequence, corresponding to the position numbers of the reference
sequence.

For example, you can also compute the number of the read sequences that align to each of the first
10 positions of the reference sequence. For this computation, use the getBaseCoverage method:

Cov = getBaseCoverage(BMObj1, 1, 10)

Cov =

1 1 2 2 3 4 4 4 5 5

Construct Sequence Alignments to a Reference Sequence

It is useful to construct and view the alignment of the read sequences that align to a specific region of
the reference sequence. It is also helpful to know which read sequences align to this region in a
BioMap object.

2-13
2 High-Throughput Sequence Analysis

For example, to retrieve the alignment of read sequences to the first 12 positions of the reference
sequence in a BioMap object, use the getAlignment method:

[Alignment_1_12, Indices] = getAlignment(BMObj2, 1, 12)

Alignment_1_12 =

CACTAGTGGCTC
CTAGTGGCTC
AGTGGCTC
GTGGCTC
GCTC

Indices =

1
2
3
4
5

Return the headers of the read sequences that align to a specific region of the reference sequence:

alignedHeaders = getHeader(BMObj2, Indices)

alignedHeaders =

'B7_591:4:96:693:509'
'EAS54_65:7:152:368:113'
'EAS51_64:8:5:734:57'
'B7_591:1:289:587:906'
'EAS56_59:8:38:671:758'

Filter Read Sequences Using SAM Flags

SAM- and BAM-formatted files include the status of 11 binary flags for each read sequence. These
flags describe different sequencing and alignment aspects of a read sequence. For more information
on the flags, see the SAM Format Specification. The filterByFlag method lets you filter the read
sequences in a BioMap object by using these flags.

Filter Unmapped Read Sequences

1 Construct a BioMap object from a SAM-formatted file.

BMObj2 = BioMap('ex1.sam');
2 Use the filterByFlag method to create a logical vector indicating the read sequences in a
BioMap object that are mapped.

LogicalVec_mapped = filterByFlag(BMObj2, 'unmappedQuery', false);

3 Use this logical vector and the getSubset method to create a new BioMap object containing
only the mapped read sequences.

filteredBMObj_1 = getSubset(BMObj2, LogicalVec_mapped);

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 Manage Sequence Read Data in Objects

Filter Read Sequences That Are Not Mapped in a Pair

1 Construct a BioMap object from a SAM-formatted file.

BMObj2 = BioMap('ex1.sam');
2 Use the filterByFlag method to create a logical vector indicating the read sequences in a
BioMap object that are mapped in a proper pair, that is, both the read sequence and its mate are
mapped to the reference sequence.

LogicalVec_paired = filterByFlag(BMObj2, 'pairedInMap', true);

3 Use this logical vector and the getSubset method to create a new BioMap object containing
only the read sequences that are mapped in a proper pair.

filteredBMObj_2 = getSubset(BMObj2, LogicalVec_paired);

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2 High-Throughput Sequence Analysis

Store and Manage Feature Annotations in Objects

In this section...
“Represent Feature Annotations in a GFFAnnotation or GTFAnnotation Object” on page 2-16
“Construct an Annotation Object” on page 2-16
“Retrieve General Information from an Annotation Object” on page 2-16
“Access Data in an Annotation Object” on page 2-17
“Use Feature Annotations with Sequence Read Data” on page 2-18

Represent Feature Annotations in a GFFAnnotation or GTFAnnotation

Object
The GFFAnnotation and GTFAnnotation objects represent a collection of feature annotations for
one or more reference sequences. You construct these objects from GFF (General Feature Format)
and GTF (Gene Transfer Format) files. Each element in the object represents a single annotation. The
properties and methods associated with the objects let you investigate and filter the data based on
reference sequence, a feature (such as CDS or exon), or a specific gene or transcript.

Construct an Annotation Object

Use the GFFAnnotation constructor function to construct a GFFAnnotation object from either a
GFF- or GTF-formatted file:

GFFAnnotObj = GFFAnnotation('tair8_1.gff')

GFFAnnotObj =

GFFAnnotation with properties:

FieldNames: {1x9 cell}

NumEntries: 3331

Use the GTFAnnotation constructor function to construct a GTFAnnotation object from a GTF-
formatted file:

GTFAnnotObj = GTFAnnotation('hum37_2_1M.gtf')

GTFAnnotObj =

GTFAnnotation with properties:

FieldNames: {1x11 cell}

NumEntries: 308

Retrieve General Information from an Annotation Object

Determine the field names and the number of entries in an annotation object by accessing the
FieldNames and NumEntries properties. For example, to see the field names for each annotation
object constructed in the previous section, query the FieldNames property:

GFFAnnotObj.FieldNames

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 Store and Manage Feature Annotations in Objects

ans =

Columns 1 through 6

'Reference' 'Start' 'Stop' 'Feature' 'Source' 'Score'

Columns 7 through 9

'Strand' 'Frame' 'Attributes'

GTFAnnotObj.FieldNames

ans =

Columns 1 through 6

'Reference' 'Start' 'Stop' 'Feature' 'Gene' 'Transcript'

Columns 7 through 11

'Source' 'Score' 'Strand' 'Frame' 'Attributes'

Determine the range of the reference sequences that are covered by feature annotations by using the
getRange method with the annotation object constructed in the previous section:

range = getRange(GFFAnnotObj)

range =

3631 498516

Access Data in an Annotation Object

Create a Structure of the Annotation Data

Creating a structure of the annotation data lets you access the field values. Use the getData method
to create a structure containing a subset of the data in a GFFAnnotation object constructed in the
previous section.

% Extract annotations for positions 1 through 10000 of the

% reference sequence
AnnotStruct = getData(GFFAnnotObj,1,10000)

AnnotStruct =

60x1 struct array with fields:

Reference
Start
Stop
Feature
Source
Score
Strand
Frame
Attributes

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2 High-Throughput Sequence Analysis

Access Field Values in the Structure

Use dot indexing to access all or specific field values in a structure.

For example, extract the start positions for all annotations:

Starts = AnnotStruct.Start;

Extract the start positions for annotations 12 through 17. Notice that you must use square brackets
when indexing a range of positions:
Starts_12_17 = [AnnotStruct(12:17).Start]

Starts_12_17 =

4706 5174 5174 5439 5439 5631

Extract the start position and the feature for the 12th annotation:
Start_12 = AnnotStruct(12).Start

Start_12 =

4706

Feature_12 = AnnotStruct(12).Feature

Feature_12 =

CDS

Use Feature Annotations with Sequence Read Data

Investigate the results of HTS sequencing experiments by using GFFAnnotation and
GTFAnnotation objects with BioMap objects. For example, you can:

• Determine counts of sequence reads aligned to regions of a reference sequence associated with
specific annotations, such as in RNA-Seq workflows.
• Find annotations within a specific range of a peak of interest in a reference sequence, such as in
ChIP-Seq workflows.

Determine Annotations of Interest

1 Construct a GTFAnnotation object from a GTF- formatted file:

GTFAnnotObj = GTFAnnotation('hum37_2_1M.gtf');
2 Use the getReferenceNames method to return the names for the reference sequences for the
annotation object:
refNames = getReferenceNames(GTFAnnotObj)

refNames =

'chr2'
3 Use the getFeatureNames method to retrieve the feature names from the annotation object:
featureNames = getFeatureNames(GTFAnnotObj)

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 Store and Manage Feature Annotations in Objects

featureNames =

'CDS'
'exon'
'start_codon'
'stop_codon'
4 Use the getGeneNames method to retrieve a list of the unique gene names from the annotation
object:
geneNames = getGeneNames(GTFAnnotObj)

geneNames =

'uc002qvu.2'
'uc002qvv.2'
'uc002qvw.2'
'uc002qvx.2'
'uc002qvy.2'
'uc002qvz.2'
'uc002qwa.2'
'uc002qwb.2'
'uc002qwc.1'
'uc002qwd.2'
'uc002qwe.3'
'uc002qwf.2'
'uc002qwg.2'
'uc002qwh.2'
'uc002qwi.3'
'uc002qwk.2'
'uc002qwl.2'
'uc002qwm.1'
'uc002qwn.1'
'uc002qwo.1'
'uc002qwp.2'
'uc002qwq.2'
'uc010ewe.2'
'uc010ewf.1'
'uc010ewg.2'
'uc010ewh.1'
'uc010ewi.2'
'uc010yim.1'

The previous steps gave us a list of available reference sequences, features, and genes associated
with the available annotations. Use this information to determine annotations of interest. For
instance, you might be interested only in annotations that are exons associated with the uc002qvv.2
gene on chromosome 2.

Filter Annotations

Use the getData method to filter the annotations and create a structure containing only the
annotations of interest, which are annotations that are exons associated with the uc002qvv.2 gene on
chromosome 2.
AnnotStruct = getData(GTFAnnotObj,'Reference','chr2',...
'Feature','exon','Gene','uc002qvv.2')

AnnotStruct =

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2 High-Throughput Sequence Analysis

12x1 struct array with fields:

Reference
Start
Stop
Feature
Gene
Transcript
Source
Score
Strand
Frame
Attributes

The return structure contains 12 elements, indicating there are 12 annotations that meet your filter
criteria.

Extract Position Ranges for Annotations of Interest

After filtering the data to include only annotations that are exons associated with the uc002qvv.2
gene on chromosome 2, use the Start and Stop fields to create vectors of the start and end positions
for the ranges associated with the 12 annotations.

StartPos = [AnnotStruct.Start];
EndPos = [AnnotStruct.Stop];

Determine Counts of Sequence Reads Aligned to Annotations

Construct a BioMap object from a BAM-formatted file containing sequence read data aligned to
chromosome 2.

BMObj3 = BioMap('ex3.bam');

Then use the range for the annotations of interest as input to the getCounts method of a BioMap
object. This returns the counts of short reads aligned to the annotations of interest.

counts = getCounts(BMObj3,StartPos,EndPos,'independent', true)

counts =

1399
1
54
221
97
125
0
1
0
65
9
12

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 Visualize and Investigate Sequence Read Alignments

Visualize and Investigate Sequence Read Alignments

In this section...
“When to Use the NGS Browser to Visualize and Investigate Data” on page 2-21
“Open the NGS Browser” on page 2-21
“Import Data into the NGS Browser” on page 2-23
“Zoom and Pan to a Specific Region of the Alignment” on page 2-25
“View Coverage of the Reference Sequence” on page 2-25
“View the Pileup View of Short Reads” on page 2-26
“Compare Alignments of Multiple Data Sets” on page 2-26
“View Location, Quality Scores, and Mapping Information” on page 2-27
“Flag Reads” on page 2-28
“Evaluate and Flag Mismatches” on page 2-28
“View Insertions and Deletions” on page 2-29
“View Feature Annotations” on page 2-29
“Print and Export the Browser Image” on page 2-30

When to Use the NGS Browser to Visualize and Investigate Data

The NGS Browser lets you visually verify and investigate the alignment of sequence reads to a
reference sequence, in support of analyses that measure genetic variations and gene expression. The
NGS Browser lets you:

• Visualize sequence reads aligned to a nucleotide reference sequence.

• Compare multiple data sets aligned against a common reference sequence.
• View coverage of different bases and regions of the reference sequence.
• Investigate quality and other details of aligned reads.
• Identify mismatches due to base-calling errors or polymorphisms.
• Visualize insertions and deletions.
• Retrieve feature annotations relative to a specific region of the reference sequence.
• Investigate regions of interest in the alignment, determined by various analyses.

You can visualize and investigate the aligned data before, during, or after any preprocessing
(filtering, quality recalibration) or analysis steps you perform on the aligned data.

Open the NGS Browser

To open the NGS Browser, type the following in the MATLAB Command Window:

ngsbrowser

Alternatively, click the NGS Browser on the Apps tab.

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2 High-Throughput Sequence Analysis

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 Visualize and Investigate Sequence Read Alignments

Import Data into the NGS Browser

Browser Displaying Reference Track, One Alignment Track, and One Annotation Track

Import a Reference Sequence

You can import a single reference sequence into the NGS Browser. The reference sequence must be
in a FASTA file.

1 Select File > Add Data from File.

2 In the Open dialog box, select a FASTA file, and then click Open.

Tip You can use the getgenbank function with the ToFile and SequenceOnly name-value pair
arguments to retrieve a reference sequence from the GenBank database and save it to a FASTA-
formatted file.

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2 High-Throughput Sequence Analysis

Import Sequence Read Alignment Data

You can import multiple data sets of sequence read alignment data. The alignment data must be in
either of the following:

• BioMap object

Tip Construct a BioMap object on page 2-8 from a SAM- or BAM-formatted file to investigate,
subset, and filter on page 2-14 the data before importing it into the NGS Browser.
• SAM- or BAM-formatted file

Note Your SAM- or BAM-formatted file must:

• Have reads ordered by start position in the reference sequence.

• Have an IDX index file (for a SAM-formatted file) or BAI and LINEARINDEX index files (for a
BAM-formatted file) stored in the same location as your source file. Otherwise, the source file
must be stored in a location to which you have write access, because MATLAB needs to create
and store index files in this location.

Tip Try using SAMtools to check if the reads in your SAM- or BAM-formatted file are ordered by
position in the reference sequence, and also to reorder them, if needed.

Tip If you do not have index files (IDX or BAI and LINEARINDEX) stored in the same location as
your source file, and your source file is stored in a location to which you do not have write access,
you cannot import data from the source file directly into the browser. Instead, construct a BioMap
object from the source file using the IndexDir name-value pair argument, and then import the
BioMap object into the browser.

To import sequence read alignment data:

1 Select File > Add Data from File or File > Import Alignment Data from MATLAB
Workspace.
2 Select a SAM-formatted file, BAM-formatted file, or BioMap object.
3 If you select a file containing multiple reference sequences, in the Select Reference dialog box,
select a reference or scan the file for available references and their mapped reads counts. Click
OK.
4 Repeat the previous steps to import additional data sets.

Import Feature Annotations

You can import multiple sets of feature annotations from GFF- or GTF-formatted files that contain
data for a single reference sequence.

1 Select File > Add Data from File.

2 In the Open dialog box, select a GFF- or GTF-formatted file, and then click Open.
3 Repeat the previous steps to import additional annotations.

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 Visualize and Investigate Sequence Read Alignments

Zoom and Pan to a Specific Region of the Alignment

To zoom in and out:

Use the toolbar buttons,

or click-drag an edge of the rubberband in the Overview area.

To pan across the alignment:

Use the toolbar buttons,

or click-drag the rubberband in the Overview area.

Tip Use the left and right arrow keys to pan in one base pair (bp) increments.

View Coverage of the Reference Sequence

At the top of each alignment track, the coverage view displays the coverage of each base in the
reference sequence. The vertical ruler on the left edge of the coverage view indicates the maximum
coverage in the display range. Hover the mouse pointer over a position in the coverage view to
display the location and counts.

Note The browser computes coverage at the base pair resolution, instead of binning, even when
zoomed out.

To change the percent coverage displayed, click anywhere in the alignment track, and then edit the
Alignment Coverage settings.

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2 High-Throughput Sequence Analysis

Tip Set Max to a value greater than 100, if needed, when comparing the coverage of multiple tracks
of reads.

View the Pileup View of Short Reads

Each alignment track includes a pileup view of the short reads aligned to the reference sequence.

Limit the depth of the reads displayed in the pileup view by setting the Maximum display read
depth in the Alignment Pileup settings.

Tip Limiting the depth of short reads in the pileup view does not change the counts displayed in the
coverage view.

Compare Alignments of Multiple Data Sets

Compare multiple data sets, with each data set in its own track, against a common reference
sequence. Use the Track List to show/hide, order, and delete tracks of data.

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 Visualize and Investigate Sequence Read Alignments

View Location, Quality Scores, and Mapping Information

Hover the mouse pointer over a position in a read to display strand direction, location, quality, and
mapping information for the base, the read, and its paired mate.

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2 High-Throughput Sequence Analysis

Flag Reads
Click anywhere in an alignment track to display the Alignment Pileup settings.

Flag Reads with Low Mapping Quality

Set the Mapping quality threshold in the Alignment Pileup section to flag low-quality reads. Reads
with a mapping quality below this level appear in a lighter shade of gray.

Flag Duplicate Reads

Select Flag duplicate reads and select an outline color.

Flag Reads with Unmapped Pairs

Select Flag reads with unmapped pair and select an outline color.

Evaluate and Flag Mismatches

Mismatches display as colored blocks or letters, depending on the zoom level.

Zoomed out view of read — Mismatches display as bars

Zoomed in view of read — Mismatches display as letters

In addition to the base Phred quality information that displays in the tooltip, you can visualize quality
differences by using the Shade mismatch bases by Phred quality settings.

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 Visualize and Investigate Sequence Read Alignments

The mismatch blocks or letters display in:

• Light shade — Mismatch bases with Phred scores below the minimum
• Graduation of medium shades — Mismatch bases with Phred scores within the minimum to
maximum range
• Dark shade — Mismatch bases with Phred scores above the maximum

View Insertions and Deletions

The NGS Browser designates insertions with a symbol. Hover the mouse pointer over the insertion
symbol to display information about it.

The NGS Browser designates deletions with dashes.

View Feature Annotations

After importing a feature annotation file, you can zoom and pan to view feature annotations
associated with a region of interest in the alignment. Hover the mouse pointer over the feature
annotation.

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Print and Export the Browser Image

Print or export the browser image by selecting File > Print Image or File > Export Image.

2-30
 Count Features from NGS Reads

Count Features from NGS Reads

This example shows how to count features from paired-end sequencing reads after aligning them to
the whole human genome curated by the Genome Reference Consortium. This example uses Genome
Reference Consortium Human Build 38 patch release 12 (GRCh38.p12) as the human genome
reference.

Prerequisites and Data Set

This example works on the UNIX® and Mac platforms only. Download the Bioinformatics Toolbox™
Interface for Bowtie Aligner support package from the Add-On Explorer.

This example assumes you have:

• Downloaded and extracted the RefSeq assembly from Genome Reference Consortium Human
Build 38 patch release 12 (GRCh38.p12).
• Downloaded and organized some paired-end reads data. This example uses the exome sequencing
data from the 1000 genomes project. Paired-end reads are indicated by '_1' and '_2' in the
filenames following the accession number. Here is one possibility for how the data can be
organized in folders:

sequence/
+--HG00096/
| +--SRR077487_1.filt.fastq
| +--SRR077487_2.filt.fastq
| +--SRR081241_1.filt.fastq
| +--SRR081241_2.filt.fastq
|
+--HG00097
+-- SRR765989_1.filt.fastq
+-- SRR765989_2.filt.fastq

Build Index

Construct an index for aligning reads to the reference using bowtie2build. The file
GCF_000001405.38_GRCh38.p12_genomic.fna contains the human reference genome in the
FASTA format. bowtieIdx is the base name of the reference index files. The '--threads 8' option
specifies the number of parallel threads to build index files faster.

bowtieIdx = 'GCF_000001405.38_GRCh38.p12_genomic.index';
buildFlag = bowtie2build('GCF_000001405.38_GRCh38.p12_genomic.fna',...
bowtieIdx,'--threads 8');

Align Reads to Reference

Use the helper function alignAllReads to align each set of paired-end reads to the reference. The
function produces a SAM file in the current folder for each sample in the 'sequence' folder.

addpath(fullfile(matlabroot,'examples','bioinfo','main')); % Make sure the supporting files are o

type alignAllReads

function samFiles = alignAllReads(indexBaseName, inputDir, outputDir)

%ALIGNALLREADS Helper function for CountFeaturesExample
%
% SAMFILES = alignAllReads(INDEXBASENAME, INPUTDIR, OUTPUTDIR) aligns

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2 High-Throughput Sequence Analysis

% paired-end sequencing reads using Bowtie2 to a prebuilt index,

% producing SAM-formatted alignment files. INDEXBASENAME
% is a character vector specifying the prefix of the index files
% created with BOWTIE2BUILD. INPUTDIR contains subdirectories for each
% sample containing paired-end reads in FASTQ format:
%
% INPUTDIR/
% +-- HG00096/
% | +-- SRR077487_1.filt.fastq
% | +-- SRR077487_2.filt.fastq
% | +-- SRR081241_1.filt.fastq
% | +-- SRR081241_2.filt.fastq
% |
% +-- HG00097/
% +-- SRR765989_1.filt.fastq
% +-- SRR765989_2.filt.fastq
%
% NOTE: each mate is distinguished by the '_1' or '_2' after the
% accession number in the filename.
%
% SAMFILES is a cell array of the resulting SAM-formatted files created
% with Bowtie2, and are placed in OUTPUTDIR.

% Copyright 2018 The MathWorks, Inc.

% Use dir() to identify sample names (subfolders of inputDir).

d = dir(inputDir);
samples = {d(3:end).name}; % skip special '.' & '..' folders
samFiles = strcat(samples, '.sam');

for s=1:numel(samples)
% Identify mate pairs of reads for each sample
sampleReadsPath = fullfile(inputDir, samples{s});
reads1 = dir([sampleReadsPath '/*_1*']);
reads2 = dir([sampleReadsPath '/*_2*']);

reads1 = fullfile(sampleReadsPath, {reads1(:).name});

reads2 = fullfile(sampleReadsPath, {reads2(:).name});

% Get full filename to SAM file

samFiles{s} = fullfile(outputDir, samFiles{s});

% Perform alignment, if file doesn't exist

if ~exist(samFiles{s}, 'file')
bowtie2(indexBaseName, reads1, reads2, samFiles{s}, '-p 2');
end
end
end

samFiles = alignAllReads(bowtieIdx,'sequence','.');

Selectively Align to Gene of Interest

SAM files can be very large. Use BioMap to select only the data for the correct reference. For this
example, consider APOE, which is a gene on Chromosome 19 linked to Alzheimer's disease. Create a
set of smaller BAM files for APOE to improve performance.

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 Count Features from NGS Reads

apoeRef = 'NC_000019.10'; % Reference name for Chromosome 19 in HG38

bamFiles = strrep(samFiles,'.sam','.bam');
for i=1:numel(samFiles)
if ~exist(bamFiles{i},'file')
bm = BioMap(samFiles{i},'SelectReference',apoeRef);
write(bm, bamFiles{i},'Format','bam');
end
end

Summarize Read Counts

Use featurecount to compare the number of transcripts for each APOE variant using a GTF file. A
full table of features is included in the GRCh38.p12 assembly in GFF format, which can be converted
to GTF using cuffgffread. This example uses a simplified GTF based on APOE transcripts.
APOE_gene.gtf is included with the software.

[FeatTable, Summary] = featurecount('APOE_gene.gtf',bamFiles,...

'Metafeature','transcript_id');

Processing GTF file APOE_gene.gtf ...

Processing BAM file ./HG00096.bam ...
Processing reference NC_000019.10 ...
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 Count Features from NGS Reads

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 Count Features from NGS Reads

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 Count Features from NGS Reads

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2 High-Throughput Sequence Analysis

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See Also
BioMap | bowtie2 | bowtie2build | cuffgffread | cufflinks | featurecount

2-40
 Identifying Differentially Expressed Genes from RNA-Seq Data

Identifying Differentially Expressed Genes from RNA-Seq Data

This example shows how to test RNA-Seq data for differentially expressed genes using a negative
binomial model.

Introduction

A typical differential expression analysis of RNA-Seq data consists of normalizing the raw counts and
performing statistical tests to reject or accept the null hypothesis that two groups of samples show no
significant difference in gene expression. This example shows how to inspect the basic statistics of
raw count data, how to determine size factors for count normalization and how to infer the most
differentially expressed genes using a negative binomial model.

The dataset for this example comprises of RNA-Seq data obtained in the experiment described by
Brooks et al. [1]. The authors investigated the effect of siRNA knock-down of pasilla, a gene known to
play an important role in the regulation of splicing in Drosophila melanogaster. The dataset consists
of 2 biological replicates of the control (untreated) samples and 2 biological replicates of the knock-
down (treated) samples.

Inspecting Read Count Tables for Genomic Features

The starting point for this analysis of RNA-Seq data is a count matrix, where the rows correspond to
genomic features of interest, the columns correspond to the given samples and the values represent
the number of reads mapped to each feature in a given sample.

The included file pasilla_count_noMM.mat contains two tables with the count matrices at the
gene level and at the exon level for each of the considered samples. You can obtain similar matrices
using the function featurecount.
load pasilla_count_noMM.mat

% preview the table of read counts for genes

geneCountTable(1:10,:)

ans =

10x6 table

ID Reference untreated3 untreated4 treated2 treated3

_______________ _________ __________ __________ ________ ________

{'FBgn0000003'} {'3R'} 0 1 1 2
{'FBgn0000008'} {'2R'} 142 117 138 132
{'FBgn0000014'} {'3R'} 20 12 10 19
{'FBgn0000015'} {'3R'} 2 4 0 1
{'FBgn0000017'} {'3L'} 6591 5127 4809 6027
{'FBgn0000018'} {'2L'} 469 530 492 574
{'FBgn0000024'} {'3R'} 5 6 10 8
{'FBgn0000028'} {'X' } 0 0 2 1
{'FBgn0000032'} {'3R'} 1160 1143 1138 1415
{'FBgn0000036'} {'3R'} 0 0 0 1

Note that when counting is performed without summarization, the individual features (exons in this
case) are reported with their metafeature assignment (genes in this case) followed by the start and
stop positions.

2-41
2 High-Throughput Sequence Analysis

% preview the table of read counts for exons

exonCountTable(1:10,:)

ans =

10x6 table

ID Reference untreated3 untreated4 treated2 tre

_________________________________ _________ __________ __________ ________ ___

{'FBgn0000003_2648220_2648518' } {'3R'} 0 0 0
{'FBgn0000008_18024938_18025756'} {'2R'} 0 1 0
{'FBgn0000008_18050410_18051199'} {'2R'} 13 9 14
{'FBgn0000008_18052282_18052494'} {'2R'} 4 2 5
{'FBgn0000008_18056749_18058222'} {'2R'} 32 27 26
{'FBgn0000008_18058283_18059490'} {'2R'} 14 18 29
{'FBgn0000008_18059587_18059757'} {'2R'} 1 4 3
{'FBgn0000008_18059821_18059938'} {'2R'} 0 0 2
{'FBgn0000015_12758093_12760298'} {'3R'} 1 2 0
{'FBgn0000017_16615461_16618374'} {'3L'} 1807 1572 1557 1

You can annotate and group the samples by creating a logical vector as follows:

samples = geneCountTable(:,3:end).Properties.VariableNames;
untreated = strncmp(samples,'untreated',length('untreated'))
treated = strncmp(samples,'treated',length('treated'))

untreated =

1x4 logical array

1 1 0 0

treated =

1x4 logical array

0 0 1 1

Plotting the Feature Assignments

The included file also contains a table geneSummaryTable with the summary of assigned and
unassigned SAM entries. You can plot the basic distribution of the counting results by considering the
number of reads that are assigned to the given genomic features (exons or genes for this example), as
well as the number of reads that are unassigned (i.e. not overlapping any feature) or ambiguous (i.e.
overlapping multiple features).

st = geneSummaryTable({'Assigned','Unassigned_ambiguous','Unassigned_noFeature'},:)
bar(table2array(st)','stacked');
legend(st.Properties.RowNames','Interpreter','none','Location','southeast');
xlabel('Sample')
ylabel('Number of reads')

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 Identifying Differentially Expressed Genes from RNA-Seq Data

st =

3x4 table

untreated3 untreated4 treated2 treated3

__________ __________ __________ __________

Assigned 1.5457e+07 1.6302e+07 1.5146e+07 1.8856e+07

Unassigned_ambiguous 1.5708e+05 1.6882e+05 1.6194e+05 1.9977e+05
Unassigned_noFeature 7.5455e+05 5.8309e+05 5.8756e+05 6.8356e+05

Note that a small fraction of the alignment records in the SAM files is not reported in the summary
table. You can notice this in the difference between the total number of records in a SAM file and the
total number of records processed during the counting procedure for that same SAM file. These
unreported records correspond to the records mapped to reference sequences that are not annotated
in the GTF file and therefore are not processed in the counting procedure. If the gene models account
for all the reference sequences used during the read mapping step, then all records are reported in
one of the categories of the summary table.

geneSummaryTable{'TotalEntries',:} - sum(geneSummaryTable{2:end,:})

ans =

2-43
2 High-Throughput Sequence Analysis

89516 95885 98207 104629

Plotting Read Coverage Across a Given Chromosome

When read counting is performed without summarization using the function featurecount, the
default IDs are composed by the attribute or metafeature (by default, gene_id) followed by the start
and the stop positions of the feature (by default, exon). You can use the exon start positions to plot
the read coverage across any chromosome in consideration, for example chromosome arm 2L.

% consider chromosome arm 2L

chr2L = strcmp(exonCountTable.Reference,'2L');
exonCount = exonCountTable{:,3:end};

% retrieve exon start positions

exonStart = regexp(exonCountTable{chr2L,1},'_(\d+)_','tokens');
exonStart = [exonStart{:}];
exonStart = cellfun(@str2num, [exonStart{:}]');

% sort exon by start positions

[~,idx] = sort(exonStart);

% plot read coverage along the genomic coordinates

figure;
plot(exonStart(idx),exonCount(idx,treated),'.-r',...
exonStart(idx),exonCount(idx,untreated),'.-b');
xlabel('Genomic position');
ylabel('Read count (exon level)');
title('Read count on Chromosome arm 2L');

% plot read coverage for each group separately

figure;
subplot(2,1,1);
plot(exonStart(idx),exonCount(idx,untreated),'.-r');
ylabel('Read count (exon level)');
title('Chromosome arm 2L (treated samples)');
subplot(2,1,2);
plot(exonStart(idx),exonCount(idx,treated),'.-b');
ylabel('Read count (exon level)');
xlabel('Genomic position');
title('Chromosome arm 2L (untreated samples)');

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Identifying Differentially Expressed Genes from RNA-Seq Data

2-45
2 High-Throughput Sequence Analysis

Alternatively, you can plot the read coverage considering the starting position of each gene in a given
chromosome. The file pasilla_geneLength.mat contains a table with the start and stop position of
each gene in the corresponding gene annotation file.

% load gene start and stop position information

load pasilla_geneLength
geneLength(1:10,:)

ans =

10x5 table

ID Name Reference Start Stop

_______________ ___________ _________ _____ _____

{'FBgn0037213'} {'CG12581'} 3R 380 10200

{'FBgn0000500'} {'Dsk' } 3R 15388 16170
{'FBgn0053294'} {'CR33294'} 3R 17136 21871
{'FBgn0037215'} {'CG12582'} 3R 23029 30295
{'FBgn0037217'} {'CG14636'} 3R 30207 41033
{'FBgn0037218'} {'aux' } 3R 37505 53244
{'FBgn0051516'} {'CG31516'} 3R 44179 45852
{'FBgn0261436'} {'DhpD' } 3R 53106 54971
{'FBgn0037220'} {'CG14641'} 3R 56475 58077
{'FBgn0015331'} {'abs' } 3R 58765 60763

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 Identifying Differentially Expressed Genes from RNA-Seq Data

% consider chromosome 3 ('Reference' is a categorical variable)

chr3 = (geneLength.Reference == '3L') | (geneLength.Reference == '3R');
sum(chr3)

% consider the counts for genes in chromosome 3

counts = geneCountTable{:,3:end};
[~,j,k] = intersect(geneCountTable{:,'ID'},geneLength{chr3,'ID'});
gstart = geneLength{k,'Start'};
gcounts = counts(j,:);

% sort according to ascending start position

[~,idx] = sort(gstart);

% plot read coverage by genomic position

figure;
plot(gstart(idx), gcounts(idx,treated),'.-r',...
gstart(idx), gcounts(idx,untreated),'.-b');
xlabel('Genomic position')
ylabel('Read count');
title('Read count on Chromosome 3');

ans =

6360

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2 High-Throughput Sequence Analysis

Normalizing Read Counts

The read count in RNA-Seq data has been found to be linearly related to the abundance of transcripts
[2]. However, the read count for a given gene depends not only on the expression level of the gene,
but also on the total number of reads sequenced and the length of the gene transcript. Therefore, in
order to infer the expression level of a gene from the read count, we need to account for the
sequencing depth and the gene transcript length. One common technique to normalize the read count
is to use the RPKM (Read Per Kilobase Mapped) values, where the read count is normalized by the
total number of reads yielded (in millions) and the length of each transcript (in kilobases). This
normalization technique, however, is not always effective since few, very highly expressed genes can
dominate the total lane count and skew the expression analysis.

A better normalization technique consists of computing the effective library size by considering a size
factor for each sample. By dividing each sample's counts by the corresponding size factors, we bring
all the count values to a common scale, making them comparable. Intuitively, if sample A is
sequenced N times deeper than sample B, the read counts of non-differentially expressed genes are
expected to be on average N times higher in sample A than in sample B, even if there is no difference
in expression.

To estimate the size factors, take the median of the ratios of observed counts to those of a pseudo-
reference sample, whose counts can be obtained by considering the geometric mean of each gene
across all samples [3]. Then, to transform the observed counts to a common scale, divide the
observed counts in each sample by the corresponding size factor.

% estimate pseudo-reference with geometric mean row by row

pseudoRefSample = geomean(counts,2);
nz = pseudoRefSample > 0;
ratios = bsxfun(@rdivide,counts(nz,:),pseudoRefSample(nz));
sizeFactors = median(ratios,1)

sizeFactors =

0.9374 0.9725 0.9388 1.1789

% transform to common scale

normCounts = bsxfun(@rdivide,counts,sizeFactors);
normCounts(1:10,:)

ans =

1.0e+03 *

0 0.0010 0.0011 0.0017

0.1515 0.1203 0.1470 0.1120
0.0213 0.0123 0.0107 0.0161
0.0021 0.0041 0 0.0008
7.0315 5.2721 5.1225 5.1124
0.5003 0.5450 0.5241 0.4869
0.0053 0.0062 0.0107 0.0068
0 0 0.0021 0.0008
1.2375 1.1753 1.2122 1.2003
0 0 0 0.0008

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 Identifying Differentially Expressed Genes from RNA-Seq Data

You can appreciate the effect of this normalization by using the function boxplot to represent
statistical measures such as median, quartiles, minimum and maximum.

figure;

subplot(2,1,1)
maboxplot(log2(counts),'title','Raw Read Count','orientation','horizontal')
ylabel('sample')
xlabel('log2(counts)')

subplot(2,1,2)
maboxplot(log2(normCounts),'title','Normalized Read Count','orientation','horizontal')
ylabel('sample')
xlabel('log2(counts)')

Computing Mean, Dispersion and Fold Change

In order to better characterize the data, we consider the mean and the dispersion of the normalized
counts. The variance of read counts is given by the sum of two terms: the variation across samples
(raw variance) and the uncertainty of measuring the expression by counting reads (shot noise or
Poisson). The raw variance term dominates for highly expressed genes, whereas the shot noise
dominates for lowly expressed genes. You can plot the empirical dispersion values against the mean
of the normalized counts in a log scale as shown below.

% consider the mean

meanTreated = mean(normCounts(:,treated),2);
meanUntreated = mean(normCounts(:,untreated),2);

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2 High-Throughput Sequence Analysis

% consider the dispersion

dispTreated = std(normCounts(:,treated),0,2) ./ meanTreated;
dispUntreated = std(normCounts(:,untreated),0,2) ./ meanUntreated;

% plot on a log-log scale

figure;
loglog(meanTreated,dispTreated,'r.');
hold on;
loglog(meanUntreated,dispUntreated,'b.');
xlabel('log2(Mean)');
ylabel('log2(Dispersion)');
legend('Treated','Untreated','Location','southwest');

Given the small number of replicates, it is not surprising to expect that the dispersion values scatter
with some variance around the true value. Some of this variance reflects sampling variance and some
reflects the true variability among the gene expressions of the samples.

You can look at the difference of the gene expression among two conditions, by calculating the fold
change (FC) for each gene, i.e. the ratio between the counts in the treated group over the counts in
the untreated group. Generally these ratios are considered in the log2 scale, so that any change is
symmetric with respect to zero (e.g. a ratio of 1/2 or 2/1 corresponds to -1 or +1 in the log scale).

% compute the mean and the log2FC

meanBase = (meanTreated + meanUntreated) / 2;
foldChange = meanTreated ./ meanUntreated;
log2FC = log2(foldChange);

2-50
 Identifying Differentially Expressed Genes from RNA-Seq Data

% plot mean vs. fold change (MA plot)

mairplot(meanTreated, meanUntreated,'Type','MA','Plotonly',true);
set(get(gca,'Xlabel'),'String','mean of normalized counts')
set(get(gca,'Ylabel'),'String','log2(fold change)')

Warning: Zero values are ignored

It is possible to annotate the values in the plot with the corresponding gene names, interactively
select genes, and export gene lists to the workspace by calling the mairplot function as illustrated
below:

mairplot(meanTreated,meanUntreated,'Labels',geneCountTable.ID,'Type','MA');

Warning: Zero values are ignored

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2 High-Throughput Sequence Analysis

It is convenient to store the information about the mean value and fold change for each gene in a
table. You can then access information about a given gene or a group of genes satisfying specific
criteria by indexing the table by gene names.

% create table with statistics about each gene

geneTable = table(meanBase,meanTreated,meanUntreated,foldChange,log2FC);
geneTable.Properties.RowNames = geneCountTable.ID;

% summary
summary(geneTable)

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 Identifying Differentially Expressed Genes from RNA-Seq Data

Variables:

meanBase: 11609x1 double

Values:

Min 0.21206
Median 201.24
Max 2.6789e+05

meanTreated: 11609x1 double

Values:

Min 0
Median 201.54
Max 2.5676e+05

meanUntreated: 11609x1 double

Values:

Min 0
Median 199.44
Max 2.7903e+05

foldChange: 11609x1 double

Values:

Min 0
Median 0.99903
Max Inf

log2FC: 11609x1 double

Values:

Min -Inf
Median -0.001406
Max Inf

% preview
geneTable(1:10,:)

ans =

10x5 table

meanBase meanTreated meanUntreated foldChange log2FC

________ ___________ _____________ __________ ___________

FBgn0000003 0.9475 1.3808 0.51415 2.6857 1.4253

FBgn0000008 132.69 129.48 135.9 0.95277 -0.069799
FBgn0000014 15.111 13.384 16.838 0.79488 -0.33119

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2 High-Throughput Sequence Analysis

FBgn0000015 1.7738 0.42413 3.1234 0.13579 -2.8806

FBgn0000017 5634.6 5117.4 6151.8 0.83186 -0.26559
FBgn0000018 514.08 505.48 522.67 0.96711 -0.048243
FBgn0000024 7.2354 8.7189 5.752 1.5158 0.60009
FBgn0000028 0.74465 1.4893 0 Inf Inf
FBgn0000032 1206.3 1206.2 1206.4 0.99983 -0.00025093
FBgn0000036 0.21206 0.42413 0 Inf Inf

% access information about a specific gene

myGene = 'FBgn0261570';
geneTable(myGene,:)
geneTable(myGene,{'meanBase','log2FC'})

% access information about a given gene list

myGeneSet = {'FBgn0261570','FBgn0261573','FBgn0261575','FBgn0261560'};
geneTable(myGeneSet,:)

ans =

1x5 table

meanBase meanTreated meanUntreated foldChange log2FC

________ ___________ _____________ __________ _______

FBgn0261570 4435.5 4939.1 3931.8 1.2562 0.32907

ans =

1x2 table

meanBase log2FC
________ _______

FBgn0261570 4435.5 0.32907

ans =

4x5 table

meanBase meanTreated meanUntreated foldChange log2FC

________ ___________ _____________ __________ _______

FBgn0261570 4435.5 4939.1 3931.8 1.2562 0.32907

FBgn0261573 2936.9 2954.8 2919.1 1.0122 0.01753
FBgn0261575 4.3776 5.6318 3.1234 1.8031 0.85047
FBgn0261560 2041.1 1494.3 2588 0.57738 -0.7924

Inferring Differential Expression with a Negative Binomial Model

Determining whether the gene expressions in two conditions are statistically different consists of
rejecting the null hypothesis that the two data samples come from distributions with equal means.
This analysis assumes the read counts are modeled according to a negative binomial distribution (as

2-54
 Identifying Differentially Expressed Genes from RNA-Seq Data

proposed in [3]). The function nbintest performs this type of hypothesis testing with three possible
options to specify the type of linkage between the variance and the mean.

By specifying the link between variance and mean as an identity, we assume the variance is equal to
the mean, and the counts are modeled by the Poisson distribution [4].

tIdentity = nbintest(counts(:,treated),counts(:,untreated),'VarianceLink','Identity');
h = plotVarianceLink(tIdentity);

% set custom title

h(1).Title.String = 'Variance Link on Treated Samples';
h(2).Title.String = 'Variance Link on Untreated Samples';

2-55
2 High-Throughput Sequence Analysis

Alternatively, by specifying the variance as the sum of the shot noise term (i.e. mean) and a constant
multiplied by the squared mean, the counts are modeled according to a distribution described in [5].
The constant term is estimated using all the rows in the data.

tConstant = nbintest(counts(:,treated),counts(:,untreated),'VarianceLink','Constant');
h = plotVarianceLink(tConstant);

% set custom title

h(1).Title.String = 'Variance Link on Treated Samples';
h(2).Title.String = 'Variance Link on Untreated Samples';

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Identifying Differentially Expressed Genes from RNA-Seq Data

2-57
2 High-Throughput Sequence Analysis

Finally, by considering the variance as the sum of the shot noise term (i.e. mean) and a locally
regressed non-parametric smooth function of the mean, the counts are modeled according to the
distribution proposed in [3].

tLocal = nbintest(counts(:,treated),counts(:,untreated),'VarianceLink','LocalRegression');
h = plotVarianceLink(tLocal);

% set custom title

h(1).Title.String = 'Variance Link on Treated Samples';
h(2).Title.String = 'Variance Link on Untreated Samples';

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Identifying Differentially Expressed Genes from RNA-Seq Data

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2 High-Throughput Sequence Analysis

In order to evaluate which fit is the best for the data in consideration, you can compare the fitting
curves in a single plot, as shown below.

h = plotVarianceLink(tLocal,'compare',true);

% set custom title

h(1).Title.String = 'Variance Link on Treated Samples';
h(2).Title.String = 'Variance Link on Untreated Samples';

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Identifying Differentially Expressed Genes from RNA-Seq Data

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2 High-Throughput Sequence Analysis

The output of nbintest includes a vector of P-values. A P-value indicates the probability that a
change in expression as strong as the one observed (or even stronger) would occur under the null
hypothesis, i.e. the conditions have no effect on gene expression. In the histogram of the P-values we
observe an enrichment of low values (due to differentially expressed genes), whereas other values are
uniformly spread (due to non-differentially expressed genes). The enrichment of values equal to 1 are
due to genes with very low counts.

figure;
histogram(tLocal.pValue,100)
xlabel('P-value')
ylabel('Frequency')
title('P-value enrichment')

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 Identifying Differentially Expressed Genes from RNA-Seq Data

Filter out those genes with relatively low count to observe a more uniform spread of non-significant P-
values across the range (0,1]. Note that this does not affect the distribution of significant P-values.

lowCountThreshold = 10;
lowCountGenes = all(counts < lowCountThreshold, 2);
histogram(tLocal.pValue(~lowCountGenes),100)
xlabel('P-value')
ylabel('Frequency')
title('P-value enrichment without low count genes')

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2 High-Throughput Sequence Analysis

Multiple Testing and Adjusted P-values

Thresholding P-values to determine what fold changes are more significant than others is not
appropriate for this type of data analysis, due to the multiple testing problem. While performing a
large number of simultaneous tests, the probability of getting a significant result simply due to
chance increases with the number of tests. In order to account for multiple testing, perform a
correction (or adjustment) of the P-values so that the probability of observing at least one significant
result due to chance remains below the desired significance level.

The Benjamini-Hochberg (BH) adjustment [6] is a statistical method that provides an adjusted P-value
answering the following question: what would be the fraction of false positives if all the genes with
adjusted P-values below a given threshold were considered significant? Set a threshold of 0.1 for the
adjusted P-values, equivalent to consider a 10% false positives as acceptable, and identify the genes
that are significantly expressed by considering all the genes with adjusted P-values below this
threshold.

% compute the adjusted P-values (BH correction)

padj = mafdr(tLocal.pValue,'BHFDR',true);

% add to the existing table

geneTable.pvalue = tLocal.pValue;
geneTable.padj = padj;

% create a table with significant genes

sig = geneTable.padj < 0.1;
geneTableSig = geneTable(sig,:);

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 Identifying Differentially Expressed Genes from RNA-Seq Data

geneTableSig = sortrows(geneTableSig,'padj');
numberSigGenes = size(geneTableSig,1)

numberSigGenes =

1904

Identifying the Most Up-regulated and Down-regulated Genes

You can now identify the most up-regulated or down-regulated genes by considering an absolute fold
change above a chosen cutoff. For example, a cutoff of 1 in log2 scale yields the list of genes that are
up-regulated with a 2 fold change.

% find up-regulated genes

up = geneTableSig.log2FC > 1;
upGenes = sortrows(geneTableSig(up,:),'log2FC','descend');
numberSigGenesUp = sum(up)

% display the top 10 up-regulated genes

top10GenesUp = upGenes(1:10,:)

% find down-regulated genes

down = geneTableSig.log2FC < -1;
downGenes = sortrows(geneTableSig(down,:),'log2FC','ascend');
numberSigGenesDown = sum(down)

% find top 10 down-regulated genes

top10GenesDown = downGenes(1:10,:)

numberSigGenesUp =

129

top10GenesUp =

10x7 table

meanBase meanTreated meanUntreated foldChange log2FC pvalue

________ ___________ _____________ __________ ______ __________

FBgn0030173 3.3979 6.7957 0 Inf Inf 0.0063115

FBgn0036822 3.1364 6.2729 0 Inf Inf 0.012203
FBgn0052548 8.158 15.269 1.0476 14.575 3.8654 0.00016945
FBgn0050495 6.8315 12.635 1.0283 12.287 3.6191 0.0018949
FBgn0063667 20.573 38.042 3.1042 12.255 3.6153 8.5037e-08
FBgn0033764 91.969 167.61 16.324 10.268 3.3601 1.8345e-25
FBgn0037290 85.845 155.46 16.228 9.5801 3.26 3.5583e-23
FBgn0033733 7.4634 13.384 1.5424 8.6773 3.1172 0.0027276
FBgn0037191 7.1766 12.753 1.6003 7.9694 2.9945 0.0047803
FBgn0033943 6.95 12.319 1.581 7.7921 2.962 0.0053635

numberSigGenesDown =

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2 High-Throughput Sequence Analysis

181

top10GenesDown =

10x7 table

meanBase meanTreated meanUntreated foldChange log2FC pvalue

________ ___________ _____________ __________ _______ _________

FBgn0053498 15.469 0 30.938 0 -Inf 9.8404e-1

FBgn0259236 6.8092 0 13.618 0 -Inf 1.5526e-0
FBgn0052500 4.3703 0 8.7405 0 -Inf 0.0006678
FBgn0039331 3.6954 0 7.3908 0 -Inf 0.001955
FBgn0040697 3.419 0 6.8381 0 -Inf 0.002737
FBgn0034972 2.9145 0 5.8291 0 -Inf 0.006856
FBgn0040967 2.6382 0 5.2764 0 -Inf 0.009603
FBgn0031923 2.3715 0 4.7429 0 -Inf 0.01616
FBgn0085359 62.473 2.9786 121.97 0.024421 -5.3557 5.5813e-3
FBgn0004854 7.4674 0.53259 14.402 0.03698 -4.7571 8.1587e-0

A good visualization of the gene expressions and their significance is given by plotting the fold
change versus the mean in log scale and coloring the data points according to the adjusted P-values.

figure
scatter(log2(geneTable.meanBase),geneTable.log2FC,3,geneTable.padj,'o')
colormap(flipud(cool(256)))
colorbar;
ylabel('log2(Fold Change)')
xlabel('log2(Mean of normalized counts)')
title('Fold change by FDR')

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 Identifying Differentially Expressed Genes from RNA-Seq Data

You can see here that for weakly expressed genes (i.e. those with low means), the FDR is generally
high because low read counts are dominated by Poisson noise and consequently any biological
variability is drowned in the uncertainties from the read counting.

References

[1] Brooks et al. Conservation of an RNA regulatory map between Drosophila and mammals. Genome
Research 2011. 21:193-202.

[2] Mortazavi et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature
Methods 2008. 5:621-628.

[3] Anders et al. Differential expression analysis for sequence count data. Genome Biology 2010.
11:R106.

[4] Marioni et al. RNA-Seq: An assessment of technical reproducibility and comparison with gene
expression arrays. Genome Research 2008. 18:1509-1517.

[5] Robinson et al. Moderated statistical test for assessing differences in tag abundance.
Bioinformatics 2007. 23(21):2881-2887.

[6] Benjamini et al. Controlling the false discovery rate: a practical and powerful approach to multiple
testing. 1995. Journal of the Royal Statistical Society, Series B57 (1):289-300.

See Also
featurecount | mairplot | nbintest | plotVarianceLink

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2 High-Throughput Sequence Analysis

More About
• “High-Throughput Sequencing”

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 Visualize NGS Data Using Genomics Viewer App

Visualize NGS Data Using Genomics Viewer App

The Genomics Viewer app lets you view and explore integrated genomic data with an embedded
version of the Integrative Genomics Viewer (IGV) [1][2]. The genomic data include NGS read
alignments, genome variants, and segmented copy number data.

The first part of this example gives a brief overview of the app and supported file formats. The second
part of the example explores a single nucleotide variation in the cytochrome p450 gene (CYP2C19).

Open the App

At the command line, type genomicsViewer. Alternatively, click the app icon on the Apps tab. The
app requires an internet connection.

By default, the app loads Human (GRCh38/hg38) as the reference sequence and Refseq Genes as the
annotation file. There are two main panels in the app. The left panel is the Tracks panel and the right
panel is the embedded IGV web application. The Track panel is a read-only area displaying the track
names, source file names, and track types. The Track panel updates accordingly as you configure the
tracks in the embedded IGV app.

The Reset button restores the app to the default view with two tracks (HG38 with Refseq Genes) and
removes any other existing tracks. Before resetting, you can save the current view as a session
(.json) file and restore it later.

Add Tracks by Importing Data

Import Reference Sequence

You can import a single reference sequence. The reference sequence must be in a FASTA file. Select
Import Reference on the Home tab. You can also import a corresponding cytoband file that contains

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2 High-Throughput Sequence Analysis

cytogenetic G-banding data. You can add local files or specify external URLs. The URL must start with
either https or gs. Other file transfer protocols, such as ftp, are not supported.

Import Sequence Read Alignment Data

You can import multiple data sets of sequence read alignment data. The alignment data must be a
BAM or CRAM file. It is not required that you have the corresponding index file (.BAI or .CRAI) in
the same location as your BAM or CRAM file. However, the absence of the index file will make the
app slower.

You can add read alignment files using Add tracks from file and Add tracks from URL options
from the Add tracks button. If you are specifying a URL, the URL must start with either https or gs.
Other file transfer protocols, such as ftp, are not supported.

Import Feature Annotations and Other Genomic Data

You can import multiple sets of feature annotations from several files that contain data for a single
reference sequence. The supported annotation files are: .BED, .GFF, .GFF3, and .GTF.

You can also import structural variants (.VCF) and visualize genetic alterations, such as insertions
and deletions.

You can view segmented copy number data (.SEG) and quantitative genomic data (.WIG, .BIGWIG,
and .BEDGRAPH), such as ChIP peaks and alignment coverage.

You can add annotation and genomic data files using Add tracks from file and Add tracks from
URL options from the Add tracks button. If you are specifying a URL, the URL must start with either
https or gs. Other file transfer protocols, such as FTP, are not supported.

Visualize Single Nucleotide Variation in Cytochrome P450

The CYP2C19 gene is a member of the cytochrome P450 gene family. Enzymes produced from
cytochrome P450 genes are involved in the metabolism of various molecules and chemicals within
cells. The CYP2C19 enzyme plays a role in the metabolizing of at least 10 percent of commonly
prescribed drugs [3]. Polymorphisms in the cytochrome p450 family may cause adverse drug
responses in individuals. One example of single nucleotide variation is rs4986893 at position
chr10:94,780,653 where G is replaced by A. This allelic variant is also known as CYP2C19*3. The
following steps show how to visualize such variation in the app using both low coverage and high
coverage data.

Load Session File

For the purposes of this example, start with a session file that has some preloaded tracks. To load the
file, click Open. Navigate to matlabroot\examples\bioinfo\, where matlabroot is the folder
where you have installed MATLAB. Select rs4986893.json.

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 Visualize NGS Data Using Genomics Viewer App

The session contains three tracks:

• Human (GRCh38/hg38) as a reference

• NA18564 as low coverage alignment data
• Refseq Genes

The low coverage alignment data comes from a female Han Chinese from Beijing, China. The sample
ID is NA18564 and the sample has been identified with the CYP2C19*3 mutation [4].

Explore Low Coverage Data

In this session file, the alignment data has been centered around the location of the mutation on the
CYP2C19 gene.

1 Click the orange bar in the coverage area to look at the position and allele distribution
information.

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2 High-Throughput Sequence Analysis

It shows that 71% of the reads have G while 29% have A at the location chr10:94,780,653. This
data is a low coverage data and may not show all the occurrences of this mutation. A high
coverage data will be explored later in the example.

Close the data tip window.

2 You can customize the various aspects of the data display in the app. For example, you can
change the track height to make more room for later tracks. Click the second gear icon. Select
Set track height. Enter 200.

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 Visualize NGS Data Using Genomics Viewer App

For details on the embedded IGV app and its available options, visit here.

Explore High Coverage Data

You can look at the high coverage data from the same sample to see the occurrences of this mutation.

1 Go to The International Genome Sample Resource website.

2 Search for the sample NA18564.
3 Download the Exome alignment file that is in the .CRAM format.
4 Also download the corresponding index file that is in the .CRAI format. Save the file in the same
location as the source .CRAM file.
5 Click the (+) icon on the Home tab. Select the downloaded .CRAM file and click Open.

The high coverage data appears as track3. You can now see many occurrences of the mutation in
several reads.
6 Click the orange bar in the coverage area to see the allele distribution. It shows that G is
replaced by A in almost 50% of the time.

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References
[1] Robinson, J., H. Thorvaldsdóttir, W. Winckler, M. Guttman, E. Lander, G. Getz, J. Mesirov. 2011.
Integrative Genomics Viewer. Nature Biotechnology. 29:24–26.

[2] Thorvaldsdóttir, H., J. Robinson, J. Mesirov. 2013. Integrative Genomics Viewer (IGV): High-
performance genomics data visualization and exploration. Briefings in Bioinformatics.
14:178–192.

[3] https://ghr.nlm.nih.gov/gene/CYP2C19

[4] https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=NA18564&Product=DNA

See Also
Genomics Viewer | Sequence Alignment | Sequence Viewer

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 3

Sequence Analysis

Sequence analysis is the process you use to find information about a nucleotide or amino acid
sequence using computational methods. Common tasks in sequence analysis are identifying genes,
determining the similarity of two genes, determining the protein coded by a gene, and determining
the function of a gene by finding a similar gene in another organism with a known function.

• “Exploring a Nucleotide Sequence Using Command Line” on page 3-2

• “Exploring a Nucleotide Sequence Using the Sequence Viewer App” on page 3-15
• “Explore a Protein Sequence Using the Sequence Viewer App” on page 3-26
• “Compare Sequences Using Sequence Alignment Algorithms” on page 3-30
• “View and Align Multiple Sequences” on page 3-43
3 Sequence Analysis

Exploring a Nucleotide Sequence Using Command Line

In this section...
“Overview of Example” on page 3-2
“Searching the Web for Sequence Information” on page 3-2
“Reading Sequence Information from the Web” on page 3-4
“Determining Nucleotide Composition” on page 3-5
“Determining Codon Composition” on page 3-8
“Open Reading Frames” on page 3-11
“Amino Acid Conversion and Composition” on page 3-13

Overview of Example
After sequencing a piece of DNA, one of the first tasks is to investigate the nucleotide content in the
sequence. Starting with a DNA sequence, this example uses sequence statistics functions to
determine mono-, di-, and trinucleotide content, and to locate open reading frames.

Searching the Web for Sequence Information

The following procedure illustrates how to use the MATLAB Help browser to search the Web for
information. In this example you are interested in studying the human mitochondrial genome. While
many genes that code for mitochondrial proteins are found in the cell nucleus, the mitochondrial has
genes that code for proteins used to produce energy.

First research information about the human mitochondria and find the nucleotide sequence for the
genome. Next, look at the nucleotide content for the entire sequence. And finally, determine open
reading frames and extract specific gene sequences.

1 Use the MATLAB Help browser to explore the Web. In the MATLAB Command Window, type

web('http://www.ncbi.nlm.nih.gov/')

A separate browser window opens with the home page for the NCBI Web site.
2 Search the NCBI Web site for information. For example, to search for the human mitochondrion
genome, from the Search list, select Genome , and in the Search list, enter mitochondrion
homo sapiens.

The NCBI Web search returns a list of links to relevant pages.

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 Exploring a Nucleotide Sequence Using Command Line

3 Select a result page. For example, click the link labeled NC_012920.

The MATLAB Help browser displays the NCBI page for the human mitochondrial genome.

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3 Sequence Analysis

Reading Sequence Information from the Web

The following procedure illustrates how to find a nucleotide sequence in a public database and read
the sequence information into the MATLAB environment. Many public databases for nucleotide
sequences are accessible from the Web. The MATLAB Command Window provides an integrated
environment for bringing sequence information into the MATLAB environment.

The consensus sequence for the human mitochondrial genome has the GenBank accession number
NC_012920. Since the whole GenBank entry is quite large and you might only be interested in the
sequence, you can get just the sequence information.

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 Exploring a Nucleotide Sequence Using Command Line

1 Get sequence information from a Web database. For example, to retrieve sequence information
for the human mitochondrial genome, in the MATLAB Command Window, type

mitochondria = getgenbank('NC_012920','SequenceOnly',true)

The getgenbank function retrieves the nucleotide sequence from the GenBank database and
creates a character array.

mitochondria =
GATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCAT
TTGGTATTTTCGTCTGGGGGGTGTGCACGCGATAGCATTGCGAGACGCTG
GAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATT
CTATTATTTATCGCACCTACGTTCAATATTACAGGCGAACATACCTACTA
AAGT . . .
2 If you don't have a Web connection, you can load the data from a MAT file included with the
Bioinformatics Toolbox software, using the command

load mitochondria

The load function loads the sequence mitochondria into the MATLAB Workspace.
3 Get information about the sequence. Type

whos mitochondria

Information about the size of the sequence displays in the MATLAB Command Window.

Name Size Bytes Class Attributes

mitochondria 1x16569 33138 char

Determining Nucleotide Composition

The following procedure illustrates how to determine the monomers and dimers, and then visualize
data in graphs and bar plots. Sections of a DNA sequence with a high percent of A+T nucleotides
usually indicate intergenic parts of the sequence, while low A+T and higher G+C nucleotide
percentages indicate possible genes. Many times high CG dinucleotide content is located before a
gene.

After you read a sequence into the MATLAB environment, you can use the sequence statistics
functions to determine if your sequence has the characteristics of a protein-coding region. This
procedure uses the human mitochondrial genome as an example. See “Reading Sequence Information
from the Web” on page 3-4.

1 Plot monomer densities and combined monomer densities in a graph. In the MATLAB Command
Window, type

ntdensity(mitochondria)

This graph shows that the genome is A+T rich.

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3 Sequence Analysis

2 Count the nucleotides using the basecount function.

basecount(mitochondria)

A list of nucleotide counts is shown for the 5'-3' strand.

ans =
A: 5124
C: 5181
G: 2169
T: 4094

3 Count the nucleotides in the reverse complement of a sequence using the seqrcomplement
function.
basecount(seqrcomplement(mitochondria))

As expected, the nucleotide counts on the reverse complement strand are complementary to the
5'-3' strand.
ans =
A: 4094
C: 2169
G: 5181
T: 5124
4 Use the function basecount with the chart option to visualize the nucleotide distribution.
figure
basecount(mitochondria,'chart','pie');

A pie chart displays in the MATLAB Figure window.

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 Exploring a Nucleotide Sequence Using Command Line

5 Count the dimers in a sequence and display the information in a bar chart.

figure
dimercount(mitochondria,'chart','bar')

ans =

AA: 1604
AC: 1495
AG: 795
AT: 1230
CA: 1534
CC: 1771
CG: 435
CT: 1440
GA: 613
GC: 711
GG: 425
GT: 419
TA: 1373
TC: 1204
TG: 513
TT: 1004

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3 Sequence Analysis

Determining Codon Composition

The following procedure illustrates how to look at codons for the six reading frames. Trinucleotides
(codon) code for an amino acid, and there are 64 possible codons in a nucleotide sequence. Knowing
the percent of codons in your sequence can be helpful when you are comparing with tables for
expected codon usage.

After you read a sequence into the MATLAB environment, you can analyze the sequence for codon
composition. This procedure uses the human mitochondria genome as an example. See “Reading
Sequence Information from the Web” on page 3-4.

1 Count codons in a nucleotide sequence. In the MATLAB Command Window, type

codoncount(mitochondria)

The codon counts for the first reading frame displays.

AAA - 167 AAC - 171 AAG - 71 AAT - 130

ACA - 137 ACC - 191 ACG - 42 ACT - 153
AGA - 59 AGC - 87 AGG - 51 AGT - 54
ATA - 126 ATC - 131 ATG - 55 ATT - 113
CAA - 146 CAC - 145 CAG - 68 CAT - 148
CCA - 141 CCC - 205 CCG - 49 CCT - 173
CGA - 40 CGC - 54 CGG - 29 CGT - 27
CTA - 175 CTC - 142 CTG - 74 CTT - 101
GAA - 67 GAC - 53 GAG - 49 GAT - 35
GCA - 81 GCC - 101 GCG - 16 GCT - 59
GGA - 36 GGC - 47 GGG - 23 GGT - 28
GTA - 43 GTC - 26 GTG - 18 GTT - 41

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 Exploring a Nucleotide Sequence Using Command Line

TAA - 157 TAC - 118 TAG - 94 TAT - 107

TCA - 125 TCC - 116 TCG - 37 TCT - 103
TGA - 64 TGC - 40 TGG - 29 TGT - 26
TTA - 96 TTC - 107 TTG - 47 TTT - 78
2 Count the codons in all six reading frames and plot the results in heat maps.
for frame = 1:3
figure
subplot(2,1,1);
codoncount(mitochondria,'frame',frame,'figure',true,...
'geneticcode','Vertebrate Mitochondrial');
title(sprintf('Codons for frame %d',frame));
subplot(2,1,2);
codoncount(mitochondria,'reverse',true,'frame',frame,...
'figure',true,'geneticcode','Vertebrate Mitochondrial');
title(sprintf('Codons for reverse frame %d',frame));
end

Heat maps display all 64 codons in the 6 reading frames.

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3 Sequence Analysis

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 Exploring a Nucleotide Sequence Using Command Line

Open Reading Frames

The following procedure illustrates how to locate the open reading frames using a specific genetic
code. Determining the protein-coding sequence for a eukaryotic gene can be a difficult task because
introns (noncoding sections) are mixed with exons. However, prokaryotic genes generally do not have
introns and mRNA sequences have the introns removed. Identifying the start and stop codons for
translation determines the protein-coding section, or open reading frame (ORF), in a sequence. Once
you know the ORF for a gene or mRNA, you can translate a nucleotide sequence to its corresponding
amino acid sequence.

After you read a sequence into the MATLAB environment, you can analyze the sequence for open
reading frames. This procedure uses the human mitochondria genome as an example. See “Reading
Sequence Information from the Web” on page 3-4.
1 Display open reading frames (ORFs) in a nucleotide sequence. In the MATLAB Command
Window, type:

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3 Sequence Analysis

seqshoworfs(mitochondria);

If you compare this output to the genes shown on the NCBI page for NC_012920, there are fewer
genes than expected. This is because vertebrate mitochondria use a genetic code slightly
different from the standard genetic code. For a list of genetic codes, see the Genetic Code table
in the aa2nt reference page.
2 Display ORFs using the Vertebrate Mitochondrial code.

orfs= seqshoworfs(mitochondria,...
'GeneticCode','Vertebrate Mitochondrial',...
'alternativestart',true);

Notice that there are now two large ORFs on the third reading frame. One starts at position 4470
and the other starts at 5904. These correspond to the genes ND2 (NADH dehydrogenase subunit
2 [Homo sapiens] ) and COX1 (cytochrome c oxidase subunit I) genes.
3 Find the corresponding stop codon. The start and stop positions for ORFs have the same indices
as the start positions in the fields Start and Stop.

ND2Start = 4470;
StartIndex = find(orfs(3).Start == ND2Start)
ND2Stop = orfs(3).Stop(StartIndex)

The stop position displays.

ND2Stop =

5511
4 Using the sequence indices for the start and stop of the gene, extract the subsequence from the
sequence.

ND2Seq = mitochondria(ND2Start:ND2Stop)

The subsequence (protein-coding region) is stored in ND2Seq and displayed on the screen.

attaatcccctggcccaacccgtcatctactctaccatctttgcaggcac
actcatcacagcgctaagctcgcactgattttttacctgagtaggcctag
aaataaacatgctagcttttattccagttctaaccaaaaaaataaaccct
cgttccacagaagctgccatcaagtatttcctcacgcaagcaaccgcatc
cataatccttc . . .
5 Determine the codon distribution.

codoncount (ND2Seq)

The codon count shows a high amount of ACC, ATA, CTA, and ATC.

AAA - 10 AAC - 14 AAG - 2 AAT - 6

ACA - 11 ACC - 24 ACG - 3 ACT - 5
AGA - 0 AGC - 4 AGG - 0 AGT - 1
ATA - 23 ATC - 24 ATG - 1 ATT - 8
CAA - 8 CAC - 3 CAG - 2 CAT - 1
CCA - 4 CCC - 12 CCG - 2 CCT - 5
CGA - 0 CGC - 3 CGG - 0 CGT - 1
CTA - 26 CTC - 18 CTG - 4 CTT - 7
GAA - 5 GAC - 0 GAG - 1 GAT - 0
GCA - 8 GCC - 7 GCG - 1 GCT - 4
GGA - 5 GGC - 7 GGG - 0 GGT - 1

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 Exploring a Nucleotide Sequence Using Command Line

GTA - 3 GTC - 2 GTG - 0 GTT - 3

TAA - 0 TAC - 8 TAG - 0 TAT - 2
TCA - 7 TCC - 11 TCG - 1 TCT - 4
TGA - 10 TGC - 0 TGG - 1 TGT - 0
TTA - 8 TTC - 7 TTG - 1 TTT - 8
6 Look up the amino acids for codons ATA, CTA, ACC, and ATC.

aminolookup('code',nt2aa('ATA'))
aminolookup('code',nt2aa('CTA'))
aminolookup('code',nt2aa('ACC'))
aminolookup('code',nt2aa('ATC'))

The following displays:

Ile isoleucine
Leu leucine
Thr threonine
Ile isoleucine

Amino Acid Conversion and Composition

The following procedure illustrates how to extract the protein-coding sequence from a gene sequence
and convert it to the amino acid sequence for the protein. Determining the relative amino acid
composition of a protein will give you a characteristic profile for the protein. Often, this profile is
enough information to identify a protein. Using the amino acid composition, atomic composition, and
molecular weight, you can also search public databases for similar proteins.

After you locate an open reading frame (ORF) in a gene, you can convert it to an amino sequence and
determine its amino acid composition. This procedure uses the human mitochondria genome as an
example. See “Open Reading Frames” on page 3-11.

1 Convert a nucleotide sequence to an amino acid sequence. In this example, only the protein-
coding sequence between the start and stop codons is converted.

ND2AASeq = nt2aa(ND2Seq,'geneticcode',...
'Vertebrate Mitochondrial')

The sequence is converted using the Vertebrate Mitochondrial genetic code. Because the
property AlternativeStartCodons is set to 'true' by default, the first codon att is
converted to M instead of I.

MNPLAQPVIYSTIFAGTLITALSSHWFFTWVGLEMNMLAFIPVLTKKMNP
RSTEAAIKYFLTQATASMILLMAILFNNMLSGQWTMTNTTNQYSSLMIMM
AMAMKLGMAPFHFWVPEVTQGTPLTSGLLLLTWQKLAPISIMYQISPSLN
VSLLLTLSILSIMAGSWGGLNQTQLRKILAYSSITHMGWMMAVLPYNPNM
TILNLTIYIILTTTAFLLLNLNSSTTTLLLSRTWNKLTWLTPLIPSTLLS
LGGLPPLTGFLPKWAIIEEFTKNNSLIIPTIMATITLLNLYFYLRLIYST
SITLLPMSNNVKMKWQFEHTKPTPFLPTLIALTTLLLPISPFMLMIL
2 Compare your conversion with the published conversion in the GenPept database.

ND2protein = getgenpept('YP_003024027','sequenceonly',true)

The getgenpept function retrieves the published conversion from the NCBI database and reads
it into the MATLAB Workspace.
3 Count the amino acids in the protein sequence.

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3 Sequence Analysis

aacount(ND2AASeq, 'chart','bar')

A bar graph displays. Notice the high content for leucine, threonine and isoleucine, and also
notice the lack of cysteine and aspartic acid.

4 Determine the atomic composition and molecular weight of the protein.

atomiccomp(ND2AASeq)
molweight (ND2AASeq)

The following displays in the MATLAB Workspace:

ans =

C: 1818
H: 2882
N: 420
O: 471
S: 25

ans =

3.8960e+004

If this sequence was unknown, you could use this information to identify the protein by
comparing it with the atomic composition of other proteins in a database.

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 Exploring a Nucleotide Sequence Using the Sequence Viewer App

Exploring a Nucleotide Sequence Using the Sequence Viewer

App
In this section...
“Overview of the Sequence Viewer” on page 3-15
“Importing a Sequence into the Sequence Viewer” on page 3-15
“Viewing Nucleotide Sequence Information” on page 3-17
“Searching for Words” on page 3-19
“Exploring Open Reading Frames” on page 3-22
“Closing the Sequence Viewer” on page 3-25

Overview of the Sequence Viewer

The Sequence Viewer integrates many of the sequence functions in the Bioinformatics Toolbox
toolbox. Instead of entering commands in the MATLAB Command Window, you can select and enter
options using the app.

Importing a Sequence into the Sequence Viewer

The first step when analyzing a nucleotide or amino acid sequence is to import sequence information
into the MATLAB environment. The Sequence Viewer can connect to Web databases such as NCBI
and EMBL and read information into the MATLAB environment.

The following procedure illustrates how to retrieve sequence information from the NCBI database on
the Web. This example uses the GenBank accession number NM_000520, which is the human gene
HEXA that is associated with Tay-Sachs disease.

Note Data in public repositories is frequently curated and updated; therefore, the results of this
example might be slightly different when you use up-to-date sequences.

1 In the MATLAB Command Window, type

seqviewer

Alternatively, click Sequence Viewer on the Apps tab.

The Sequence Viewer opens without a sequence loaded. Notice that the panes to the right and
bottom are blank.
2 To retrieve a sequence from the NCBI database, select File > Download Sequence from >
NCBI.

The Download Sequence from NCBI dialog box opens.

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3 Sequence Analysis

3 In the Enter Sequence box, type an accession number for an NCBI database entry, for example,
NM_000520. Click the Nucleotide option button, and then click OK.

The MATLAB software accesses the NCBI database on the Web, loads nucleotide sequence
information for the accession number you entered, and calculates some basic statistics.

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 Exploring a Nucleotide Sequence Using the Sequence Viewer App

Viewing Nucleotide Sequence Information

After you import a sequence into the Sequence Viewer app, you can read information stored with
the sequence, or you can view graphic representations for ORFs and CDSs.

1 In the left pane tree, click Comments. The right pane displays general information about the
sequence.

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3 Sequence Analysis

2 Now click Features. The right pane displays NCBI feature information, including index numbers
for a gene and any CDS sequences.
3 Click ORF to show the search results for ORFs in the six reading frames.

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 Exploring a Nucleotide Sequence Using the Sequence Viewer App

4 Click Annotated CDS to show the protein coding part of a nucleotide sequence.

Searching for Words

You can also search for characteristic words or sequence patterns using regular expressions. You can
enter the IUB/IUPAC nucleotide and amino acid symbols that are automatically converted to
corresponding nucleotides and amino acids accordingly. For details about how symbols are
interpreted, see the Nucleotide Conversion and Amino Acid Conversion tables of seq2regexp.

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3 Sequence Analysis

For instance, if you search for the word 'TAR' with the Regular Expression box checked, the app
highlights all the occurrences of 'TAA' and 'TAG' in the sequence since R = [AG].

1 Select Sequence > Find Word.

2 In the Find Word dialog box, type a sequence word or pattern, for example, atg, and then click
Find.

The Sequence Viewer searches and displays the location of the selected word.

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 Exploring a Nucleotide Sequence Using the Sequence Viewer App

3
Clear the display by clicking the Clear Word Selection button on the toolbar.

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3 Sequence Analysis

Exploring Open Reading Frames

The following procedure illustrates how to identify the protein coding part of a nucleotide sequence
and copy it into a new view. Identifying coding sections of a nucleotide sequence is a common
bioinformatics task. After locating the coding part of a sequence, you can copy it to a new view,
translate it to an amino acid sequence, and continue with your analysis.

1 In the left pane, click ORF.

The Sequence Viewer displays the ORFs for the six reading frames in the lower-right pane.
Hover the cursor over a frame to display information about it.

2 Click the longest ORF on reading frame 2.

The ORF is highlighted to indicate the part of the sequence that is selected.

3 Right-click the selected ORF and then select Export to Workspace. In the Export to MATLAB
Workspace dialog box, type a variable name, for example, NM_000520_ORF_2, then click
Export.

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 Exploring a Nucleotide Sequence Using the Sequence Viewer App

The NM_000520_ORF_2 variable is added to the MATLAB Workspace.

4 Select File > Import from Workspace. Type the name of a variable with an exported ORF, for
example, NM_000520_ORF_2, and then click Import.

The Sequence Viewer adds a tab at the bottom for the new sequence while leaving the original
sequence open.

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3 Sequence Analysis

5 In the left pane, click Full Translation. Select Display > Amino Acid Residue Display > One
Letter Code.

The Sequence Viewer displays the amino acid sequence below the nucleotide sequence.

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 Exploring a Nucleotide Sequence Using the Sequence Viewer App

Closing the Sequence Viewer

Close the Sequence Viewer from the MATLAB command line using the following syntax:

seqviewer('close')

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3 Sequence Analysis

Explore a Protein Sequence Using the Sequence Viewer App

In this section...
“Overview of the Sequence Viewer” on page 3-26
“Viewing Amino Acid Sequence Statistics” on page 3-26
“Closing the Sequence Viewer” on page 3-28
“References” on page 3-29

Overview of the Sequence Viewer

The Sequence Viewer integrates many of the sequence functions in the Bioinformatics Toolbox
toolbox. Instead of entering commands in the MATLAB Command Window, you can select and enter
options using the app.

Viewing Amino Acid Sequence Statistics

The following procedure illustrates how to view an amino acid sequence for an ORF located in a
nucleotide sequence. You can import your own amino acid sequence, or you can get a protein
sequence from the GenBank database. This example uses the GenBank accession number
NP_000511.1, which is the alpha subunit for a human enzyme associated with Tay-Sachs disease.

1 Select File > Download Sequence from > NCBI.

The Download Sequence from NCBI dialog box opens.

2 In the Enter Sequence box, type an accession number for an NCBI database entry, for example,
NP_000511.1. Click the Protein option button, and then click OK.

The Sequence Viewer accesses the NCBI database on the Web and loads amino acid sequence
information for the accession number you entered.

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 Explore a Protein Sequence Using the Sequence Viewer App

3 Select Display > Amino Acid Color Scheme, and then select Charge, Function,
Hydrophobicity, Structure, or Taylor. For example, select Function.

The display colors change to highlight charge information about the amino acid residues. The
following table shows color legends for the amino acid color schemes.

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3 Sequence Analysis

Amino Acid Color Scheme Color Legend

Charge • Acidic — Red
• Basic — Light Blue
• Neutral — Black
Function • Acidic — Red
• Basic — Light Blue
• Hydropobic, nonpolar — Black
• Polar, uncharged — Green
Hydrophobicity • Hydrophilic — Light Blue
• Hydrophobic — Black
Structure • Ambivalent — Dark Green
• External — Light Blue
• Internal — Orange
Taylor Each amino acid is assigned its own color, based on the
colors proposed by W.R. Taylor on page 3-29.

Closing the Sequence Viewer

Close the Sequence Viewer from the MATLAB command line using the following syntax:

seqviewer('close')

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 Explore a Protein Sequence Using the Sequence Viewer App

References

[1] Taylor, W.R. (1997). Residual colours: a proposal for aminochromography. Protein Engineering 10,
7, 743–746.

3-29
3 Sequence Analysis

Compare Sequences Using Sequence Alignment Algorithms

In this section...
“Overview of Example” on page 3-30
“Find a Model Organism to Study” on page 3-30
“Retrieve Sequence Information from a Public Database” on page 3-31
“Search a Public Database for Related Genes” on page 3-33
“Locate Protein Coding Sequences” on page 3-34
“Compare Amino Acid Sequences” on page 3-36

Overview of Example
Determining the similarity between two sequences is a common task in computational biology.
Starting with a nucleotide sequence for a human gene, this example uses alignment algorithms to
locate and verify a corresponding gene in a model organism.

Find a Model Organism to Study

In this example, you are interested in studying Tay-Sachs disease. Tay-Sachs is an autosomal
recessive disease caused by the absence of the enzyme beta-hexosaminidase A (Hex A). This enzyme
is responsible for the breakdown of gangliosides (GM2) in brain and nerve cells.

First, research information about Tay-Sachs and the enzyme that is associated with this disease, then
find the nucleotide sequence for the human gene that codes for the enzyme, and finally find a
corresponding gene in another organism to use as a model for study.

1 Use the MATLAB Help browser to explore the Web. In the MATLAB Command window, type

web('http://www.ncbi.nlm.nih.gov/books/NBK22250/')

The MATLAB Help browser opens with the Tay-Sachs disease page in the Genes and Diseases
section of the NCBI web site. This section provides a comprehensive introduction to medical
genetics. In particular, this page contains an introduction and pictorial representation of the
enzyme Hex A and its role in the metabolism of the lipid GM2 ganglioside.

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 Compare Sequences Using Sequence Alignment Algorithms

2 After completing your research, you have concluded the following:

The gene HEXA codes for the alpha subunit of the dimer enzyme hexosaminidase A (Hex A),
while the gene HEXB codes for the beta subunit of the enzyme. A third gene, GM2A, codes for
the activator protein GM2. However, it is a mutation in the gene HEXA that causes Tay-Sachs.

Retrieve Sequence Information from a Public Database

The following procedure illustrates how to find the nucleotide sequence for a human gene in a public
database and read the sequence information into the MATLAB environment. Many public databases
for nucleotide sequences (for example, GenBank, EMBL-EBI) are accessible from the Web. The
MATLAB Command Window with the MATLAB Help browser provide an integrated environment for
searching the Web and bringing sequence information into the MATLAB environment.

After you locate a sequence, you need to move the sequence data into the MATLAB Workspace.

1 Open the MATLAB Help browser to the NCBI Web site. In the MATLAB Command Widow, type
web('http://www.ncbi.nlm.nih.gov/')

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3 Sequence Analysis

The MATLAB Help browser window opens with the NCBI home page.
2 Search for the gene you are interested in studying. For example, from the Search list, select
Nucleotide, and in the for box enter Tay-Sachs.

The search returns entries for the genes that code the alpha and beta subunits of the enzyme
hexosaminidase A (Hex A), and the gene that codes the activator enzyme. The NCBI reference for
the human gene HEXA has accession number NM_000520.

3 Get sequence data into the MATLAB environment. For example, to get sequence information for
the human gene HEXA, type

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 Compare Sequences Using Sequence Alignment Algorithms

humanHEXA = getgenbank('NM_000520')

Note Blank spaces in GenBank accession numbers use the underline character. Entering 'NM
00520' returns the wrong entry.

The human gene is loaded into the MATLAB Workspace as a structure.

humanHEXA =

LocusName: 'NM_000520'
LocusSequenceLength: '2255'
LocusNumberofStrands: ''
LocusTopology: 'linear'
LocusMoleculeType: 'mRNA'
LocusGenBankDivision: 'PRI'
LocusModificationDate: '13-AUG-2006'
Definition: 'Homo sapiens hexosaminidase A (alpha polypeptide) (HEXA), mRNA.'
Accession: 'NM_000520'
Version: 'NM_000520.2'
GI: '13128865'
Project: []
Keywords: []
Segment: []
Source: 'Homo sapiens (human)'
SourceOrganism: [4x65 char]
Reference: {1x58 cell}
Comment: [15x67 char]
Features: [74x74 char]
CDS: [1x1 struct]
Sequence: [1x2255 char]
SearchURL: [1x108 char]
RetrieveURL: [1x97 char]

Search a Public Database for Related Genes

The following procedure illustrates how to find the nucleotide sequence for a mouse gene related to a
human gene, and read the sequence information into the MATLAB environment. The sequence and
function of many genes is conserved during the evolution of species through homologous genes.
Homologous genes are genes that have a common ancestor and similar sequences. One goal of
searching a public database is to find similar genes. If you are able to locate a sequence in a database
that is similar to your unknown gene or protein, it is likely that the function and characteristics of the
known and unknown genes are the same.

After finding the nucleotide sequence for a human gene, you can do a BLAST search or search in the
genome of another organism for the corresponding gene. This procedure uses the mouse genome as
an example.

1 Open the MATLAB Help browser to the NCBI Web site. In the MATLAB Command window, type

web('http://www.ncbi.nlm.nih.gov')
2 Search the nucleotide database for the gene or protein you are interested in studying. For
example, from the Search list, select Nucleotide, and in the for box enter hexosaminidase
A.

The search returns entries for the mouse and human genomes. The NCBI reference for the
mouse gene HEXA has accession number AK080777.

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3 Sequence Analysis

3 Get sequence information for the mouse gene into the MATLAB environment. Type

mouseHEXA = getgenbank('AK080777')

The mouse gene sequence is loaded into the MATLAB Workspace as a structure.

mouseHEXA =

LocusName: 'AK080777'
LocusSequenceLength: '1839'
LocusNumberofStrands: ''
LocusTopology: 'linear'
LocusMoleculeType: 'mRNA'
LocusGenBankDivision: 'HTC'
LocusModificationDate: '02-SEP-2005'
Definition: [1x150 char]
Accession: 'AK080777'
Version: 'AK080777.1'
GI: '26348756'
Project: []
Keywords: 'HTC; CAP trapper.'
Segment: []
Source: 'Mus musculus (house mouse)'
SourceOrganism: [4x65 char]
Reference: {1x8 cell}
Comment: [8x66 char]
Features: [33x74 char]
CDS: [1x1 struct]
Sequence: [1x1839 char]
SearchURL: [1x107 char]
RetrieveURL: [1x97 char]

Locate Protein Coding Sequences

The following procedure illustrates how to convert a sequence from nucleotides to amino acids and
identify the open reading frames. A nucleotide sequence includes regulatory sequences before and
after the protein coding section. By analyzing this sequence, you can determine the nucleotides that
code for the amino acids in the final protein.

After you have a list of genes you are interested in studying, you can determine the protein coding
sequences. This procedure uses the human gene HEXA and mouse gene HEXA as an example.

1 If you did not retrieve gene data from the Web, you can load example data from a MAT-file
included with the Bioinformatics Toolbox software. In the MATLAB Command window, type

load hexosaminidase

The structures humanHEXA and mouseHEXA load into the MATLAB Workspace.

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 Compare Sequences Using Sequence Alignment Algorithms

2 Locate open reading frames (ORFs) in the human gene. For example, for the human gene HEXA,
type
humanORFs = seqshoworfs(humanHEXA.Sequence)

seqshoworfs creates the output structure humanORFs. This structure contains the position of
the start and stop codons for all open reading frames (ORFs) on each reading frame.
humanORFs =

1x3 struct array with fields:

Start
Stop

The Help browser opens displaying the three reading frames with the ORFs colored blue, red,
and green. Notice that the longest ORF is in the first reading frame.

3-35
3 Sequence Analysis

3 Locate open reading frames (ORFs) in the mouse gene. Type:

mouseORFs = seqshoworfs(mouseHEXA.Sequence)

seqshoworfs creates the structure mouseORFS.

mouseORFs =

1x3 struct array with fields:

Start
Stop

The mouse gene shows the longest ORF on the first reading frame.

Compare Amino Acid Sequences

The following procedure illustrates how to use global and local alignment functions to compare two
amino acid sequences. You could use alignment functions to look for similarities between two
nucleotide sequences, but alignment functions return more biologically meaningful results when you
are using amino acid sequences.

After you have located the open reading frames on your nucleotide sequences, you can convert the
protein coding sections of the nucleotide sequences to their corresponding amino acid sequences,
and then you can compare them for similarities.

1 Using the open reading frames identified previously, convert the human and mouse DNA
sequences to the amino acid sequences. Because both the human and mouse HEXA genes were in
the first reading frames (default), you do not need to indicate which frame. Type

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 Compare Sequences Using Sequence Alignment Algorithms

humanProtein = nt2aa(humanHEXA.Sequence);
mouseProtein = nt2aa(mouseHEXA.Sequence);
2 Draw a dot plot comparing the human and mouse amino acid sequences. Type

seqdotplot(mouseProtein,humanProtein,4,3)
ylabel('Mouse hexosaminidase A (alpha subunit)')
xlabel('Human hexosaminidase A (alpha subunit)')

Dot plots are one of the easiest ways to look for similarity between sequences. The diagonal line
shown below indicates that there may be a good alignment between the two sequences.

3 Globally align the two amino acid sequences, using the Needleman-Wunsch algorithm. Type

[GlobalScore, GlobalAlignment] = nwalign(humanProtein,...

mouseProtein);
showalignment(GlobalAlignment)

showalignment displays the global alignment of the two sequences in the Help browser. Notice
that the calculated identity between the two sequences is 60%.

3-37
3 Sequence Analysis

The alignment is very good between amino acid position 69 and 599, after which the two
sequences appear to be unrelated. Notice that there is a stop (*) in the sequence at this point. If
you shorten the sequences to include only the amino acids that are in the protein you might get a
better alignment. Include the amino acid positions from the first methionine (M) to the first stop
(*) that occurs after the first methionine.
4 Trim the sequence from the first start amino acid (usually M) to the first stop (*) and then try
alignment again. Find the indices for the stops in the sequences.

3-38
 Compare Sequences Using Sequence Alignment Algorithms

humanStops = find(humanProtein == '*')

humanStops =

41 599 611 713 722 730

mouseStops = find(mouseProtein == '*')

mouseStops =

539 557 574 606

Looking at the amino acid sequence for humanProtein, the first M is at position 70, and the first
stop after that position is actually the second stop in the sequence (position 599). Looking at the
amino acid sequence for mouseProtein, the first M is at position 11, and the first stop after that
position is the first stop in the sequence (position 557).
5 Truncate the sequences to include only amino acids in the protein and the stop.

humanProteinORF = humanProtein(70:humanStops(2))

humanProteinORF =

MTSSRLWFSLLLAAAFAGRATALWPWPQNFQTSDQRYVLYPNNFQFQYDV
SSAAQPGCSVLDEAFQRYRDLLFGSGSWPRPYLTGKRHTLEKNVLVVSVV
TPGCNQLPTLESVENYTLTINDDQCLLLSETVWGALRGLETFSQLVWKSA
EGTFFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVMAYNKLNV
FHWHLVDDPSFPYESFTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRG
IRVLAEFDTPGHTLSWGPGIPGLLTPCYSGSEPSGTFGPVNPSLNNTYEF
MSTFFLEVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQ
LESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDIPVNY
MKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYIVEPLAFEGTPEQKA
LVIGGEACMWGEYVDNTNLVPRLWPRAGAVAERLWSNKLTSDLTFAYERL
SHFRCELLRRGVQAQPLNVGFCEQEFEQT*

mouseProteinORF = mouseProtein(11:mouseStops(1))

mouseProteinORF =

MAGCRLWVSLLLAAALACLATALWPWPQYIQTYHRRYTLYPNNFQFRYHV
SSAAQAGCVVLDEAFRRYRNLLFGSGSWPRPSFSNKQQTLGKNILVVSVV
TAECNEFPNLESVENYTLTINDDQCLLASETVWGALRGLETFSQLVWKSA
EGTFFINKTKIKDFPRFPHRGVLLDTSRHYLPLSSILDTLDVMAYNKFNV
FHWHLVDDSSFPYESFTFPELTRKGSFNPVTHIYTAQDVKEVIEYARLRG
IRVLAEFDTPGHTLSWGPGAPGLLTPCYSGSHLSGTFGPVNPSLNSTYDF
MSTLFLEISSVFPDFYLHLGGDEVDFTCWKSNPNIQAFMKKKGFTDFKQL
ESFYIQTLLDIVSDYDKGYVVWQEVFDNKVKVRPDTIIQVWREEMPVEYM
LEMQDITRAGFRALLSAPWYLNRVKYGPDWKDMYKVEPLAFHGTPEQKAL
VIGGEACMWGEYVDSTNLVPRLWPRAGAVAERLWSSNLTTNIDFAFKRLS
HFRCELVRRGIQAQPISVGCCEQEFEQT*
6 Globally align the trimmed amino acid sequences. Type

[GlobalScore_trim, GlobalAlignment_trim] = nwalign(humanProteinORF,...

mouseProteinORF);
showalignment(GlobalAlignment_trim)

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3 Sequence Analysis

showalignment displays the results for the second global alignment. Notice that the percent
identity for the untrimmed sequences is 60% and 84% for trimmed sequences.

7 Another way to truncate an amino acid sequence to only those amino acids in the protein is to
first truncate the nucleotide sequence with indices from the seqshoworfs function. Remember
that the ORF for the human HEXA gene and the ORF for the mouse HEXA were both on the first
reading frame.

humanORFs = seqshoworfs(humanHEXA.Sequence)

humanORFs =

1x3 struct array with fields:

Start
Stop

3-40
 Compare Sequences Using Sequence Alignment Algorithms

mouseORFs = seqshoworfs(mouseHEXA.Sequence)

mouseORFs =

1x3 struct array with fields:

Start
Stop

humanPORF = nt2aa(humanHEXA.Sequence(humanORFs(1).Start(1):...
humanORFs(1).Stop(1)));

mousePORF = nt2aa(mouseHEXA.Sequence(mouseORFs(1).Start(1):...
mouseORFs(1).Stop(1)));

[GlobalScore2, GlobalAlignment2] = nwalign(humanPORF, mousePORF);

Show the alignment in the Help browser.

showalignment(GlobalAlignment2)

The result from first truncating a nucleotide sequence before converting it to an amino acid
sequence is the same as the result from truncating the amino acid sequence after conversion.
See the result in step 6.

An alternative method to working with subsequences is to use a local alignment function with the
nontruncated sequences.
8 Locally align the two amino acid sequences using a Smith-Waterman algorithm. Type

[LocalScore, LocalAlignment] = swalign(humanProtein,...

mouseProtein)

LocalScore =
1057

LocalAlignment =

RGDQR-AMTSSRLWFSLLLAAAFAGRATALWPWPQNFQTSDQRYV . . .
|| | ||:: ||| |||||||:| ||||||||| :|| :||: . . .
RGAGRWAMAGCRLWVSLLLAAALACLATALWPWPQYIQTYHRRYT . . .
9 Show the alignment in color.

showalignment(LocalAlignment)

3-41
3 Sequence Analysis

3-42
 View and Align Multiple Sequences

View and Align Multiple Sequences

In this section...
“Overview of the Sequence Alignment App” on page 3-43
“Visualize Multiple Sequence Alignment” on page 3-43
“Adjust Sequence Alignments Manually” on page 3-44
“Rearrange Rows” on page 3-52
“Generate Phylogenetic Tree from Aligned Sequences” on page 3-54

Overview of the Sequence Alignment App

The Sequence Alignment app integrates many sequence and multiple alignment functions in the
toolbox. Instead of entering commands in the MATLAB Command Window, you can use this app to
visually inspect a multiple alignment and make manual adjustments.

Visualize Multiple Sequence Alignment

1 Read a multiple sequence alignment file of the gag polyprotein for several HIV strains.

gagaa = multialignread('aagag.aln')
2 View the aligned sequences in the Sequence Alignment app.

seqalignviewer(gagaa);

3-43
3 Sequence Analysis

Adjust Sequence Alignments Manually

Algorithms for aligning multiple sequences do not always produce an optimal result. By visually
inspecting the alignment, you can identify areas whose alignment can be improved by a manual
adjustment.

1 To better visualize the sequence alignments, you can zoom in by selecting Display > Zoom in.
Select this option multiple times until you achieve the zoom level you want.
2 Identify an area where you could improve the alignment.

3-44
 View and Align Multiple Sequences

3 Click a letter or a region. The selected region is the center block. You can then drag the
sequence(s) to the left or right of the center block.

3-45
3 Sequence Analysis

4 To move a single letter (T in this example), click and drag the letter T (center block) to the right
to insert a gap.

3-46
 View and Align Multiple Sequences

5 Close the gap by dragging the letter back to the left.

3-47
3 Sequence Analysis

6 You can also move multiple residues (a subsequence). Suppose you want to move a subsequence
to available gaps. First select the gap region that you want to fill in.

3-48
 View and Align Multiple Sequences

7 Drag the subsequence(s) from the right or left of the gap region into the gap area.

3-49
3 Sequence Analysis

8 Suppose you want to remove one or more of the aligned sequences. First select the sequence(s)
to be removed. Then select Edit > Delete Sequences.

3-50
 View and Align Multiple Sequences

9 Remove empty columns by selecting Edit > Remove Empty Columns.

3-51
3 Sequence Analysis

10 After the edit, you can export the aligned sequences or consensus sequence to a FASTA file or
MATLAB Workspace from the File menu.

Rearrange Rows
You can move the rows (sequences) up or down by one row. You can also move selected rows to the
top or bottom of the list.

3-52
 View and Align Multiple Sequences

The selected sequence moves to the bottom of the list.

3-53
3 Sequence Analysis

Generate Phylogenetic Tree from Aligned Sequences

You can generate a phylogenetic tree using the aligned sequences from within the app. You can select
a subset of sequences or use all the sequences to generate a tree.

Select Display > View Tree > Selected... to generate a tree from selected sequences.

3-54
 View and Align Multiple Sequences

A phylogenetic tree for the sequences is displayed in the Phylogenetic Tree app. For details on the
app, see “Using the Phylogenetic Tree App” on page 5-2.

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3 Sequence Analysis

See Also
NGS Browser | Sequence Alignment | Sequence Viewer | seqalignviewer

More About
• “Sequence Alignments” on page 1-7
• “Aligning Pairs of Sequences”

3-56
 4

Microarray Analysis

• “Managing Gene Expression Data in Objects” on page 4-2

• “Representing Expression Data Values in DataMatrix Objects” on page 4-5
• “Representing Expression Data Values in ExptData Objects” on page 4-9
• “Representing Sample and Feature Metadata in MetaData Objects” on page 4-12
• “Representing Experiment Information in a MIAME Object” on page 4-16
• “Representing All Data in an ExpressionSet Object” on page 4-19
• “Visualizing Microarray Images” on page 4-23
4 Microarray Analysis

Managing Gene Expression Data in Objects

Microarray gene expression experiments are complex, containing data and information from various
sources. The data and information from such an experiment is typically subdivided into four
categories:

• Measured expression data values

• Sample metadata
• Microarray feature metadata
• Descriptions of experiment methods and conditions

In MATLAB, you can represent all the previous data and information in an ExpressionSet object,
which typically contains the following objects:

• One ExptData object containing expression values from a microarray experiment in one or more
DataMatrix objects
• One MetaData object containing sample metadata in two dataset arrays
• One MetaData object containing feature metadata in two dataset arrays
• One MIAME object containing experiment descriptions

The following graphic illustrates a typical ExpressionSet object and its component objects.

4-2
 Managing Gene Expression Data in Objects

Each element (DataMatrix object) in the ExpressionSet object has an element name. Also, there is
always one DataMatrix object whose element name is Expressions.

An ExpressionSet object lets you store, manage, and subset the data from a microarray gene
expression experiment. An ExpressionSet object includes properties and methods that let you access,
retrieve, and change data, metadata, and other information about the microarray experiment. These
properties and methods are useful to view and analyze the data. For a list of the properties and
methods, see ExpressionSet class.

To learn more about constructing and using objects for microarray gene expression data and
information, see:

• “Representing Expression Data Values in DataMatrix Objects” on page 4-5

• “Representing Expression Data Values in ExptData Objects” on page 4-9

4-3
4 Microarray Analysis

• “Representing Sample and Feature Metadata in MetaData Objects” on page 4-12

• “Representing Experiment Information in a MIAME Object” on page 4-16
• “Representing All Data in an ExpressionSet Object” on page 4-19

4-4
 Representing Expression Data Values in DataMatrix Objects

Representing Expression Data Values in DataMatrix Objects

In this section...
“Overview of DataMatrix Objects” on page 4-5
“Constructing DataMatrix Objects” on page 4-5
“Getting and Setting Properties of a DataMatrix Object” on page 4-6
“Accessing Data in DataMatrix Objects” on page 4-6

Overview of DataMatrix Objects

The toolbox includes functions, objects, and methods for creating, storing, and accessing microarray
data.

The object constructor function, DataMatrix, lets you create a DataMatrix object to encapsulate
data and metadata (row and column names) from a microarray experiment. A DataMatrix object
stores experimental data in a matrix, with rows typically corresponding to gene names or probe
identifiers, and columns typically corresponding to sample identifiers. A DataMatrix object also stores
metadata, including the gene names or probe identifiers (as the row names) and sample identifiers
(as the column names).

You can reference microarray expression values in a DataMatrix object the same way you reference
data in a MATLAB array, that is, by using linear or logical indexing. Alternately, you can reference this
experimental data by gene (probe) identifiers and sample identifiers. Indexing by these identifiers lets
you quickly and conveniently access subsets of the data without having to maintain additional index
arrays.

Many MATLAB operators and arithmetic functions are available to DataMatrix objects by means of
methods. These methods let you modify, combine, compare, analyze, plot, and access information
from DataMatrix objects. Additionally, you can easily extend the functionality by using general
element-wise functions, dmarrayfun and dmbsxfun, and by manually accessing the properties of a
DataMatrix object.

Note For tables describing the properties and methods of a DataMatrix object, see the DataMatrix
object reference page.

Constructing DataMatrix Objects

1 Load the MAT-file, provided with the Bioinformatics Toolbox software, that contains yeast data.
This MAT-file includes three variables: yeastvalues, a 614-by-7 matrix of gene expression data,
genes, a cell array of 614 GenBank accession numbers for labeling the rows in yeastvalues,
and times, a 1-by-7 vector of time values for labeling the columns in yeastvalues.

load filteredyeastdata
2 Create variables to contain a subset of the data, specifically the first five rows and first four
columns of the yeastvalues matrix, the genes cell array, and the times vector.

yeastvalues = yeastvalues(1:5,1:4);
genes = genes(1:5,:);
times = times(1:4);

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4 Microarray Analysis

3 Import the microarray object package so that the DataMatrix constructor function will be
available.

import bioma.data.*
4 Use the DataMatrix constructor function to create a small DataMatrix object from the gene
expression data.

dmo = DataMatrix(yeastvalues,genes,times)

dmo =

0 9.5 11.5 13.5

SS DNA -0.131 1.699 -0.026 0.365
YAL003W 0.305 0.146 -0.129 -0.444
YAL012W 0.157 0.175 0.467 -0.379
YAL026C 0.246 0.796 0.384 0.981
YAL034C -0.235 0.487 -0.184 -0.669

Getting and Setting Properties of a DataMatrix Object

You use the get and set methods to retrieve and set properties of a DataMatrix object.

1 Use the get method to display the properties of the DataMatrix object, dmo.

get(dmo)
Name: ''
RowNames: {5x1 cell}
ColNames: {' 0' ' 9.5' '11.5' '13.5'}
NRows: 5
NCols: 4
NDims: 2
ElementClass: 'double'
2 Use the set method to specify a name for the DataMatrix object, dmo.

dmo = set(dmo,'Name','MyDMObject');
3 Use the get method again to display the properties of the DataMatrix object, dmo.

get(dmo)
Name: 'MyDMObject'
RowNames: {5x1 cell}
ColNames: {' 0' ' 9.5' '11.5' '13.5'}
NRows: 5
NCols: 4
NDims: 2
ElementClass: 'double'

Note For a description of all properties of a DataMatrix object, see the DataMatrix object reference
page.

Accessing Data in DataMatrix Objects

DataMatrix objects support the following types of indexing to extract, assign, and delete data:

4-6
 Representing Expression Data Values in DataMatrix Objects

• Parenthesis ( ) indexing
• Dot . indexing

Parentheses () Indexing

Use parenthesis indexing to extract a subset of the data in dmo and assign it to a new DataMatrix
object dmo2:

dmo2 = dmo(1:5,2:3)
dmo2 =
9.5 11.5
SS DNA 1.699 -0.026
YAL003W 0.146 -0.129
YAL012W 0.175 0.467
YAL026C 0.796 0.384
YAL034C 0.487 -0.184

Use parenthesis indexing to extract a subset of the data using row names and column names, and
assign it to a new DataMatrix object dmo3:

dmo3 = dmo({'SS DNA','YAL012W','YAL034C'},'11.5')

dmo3 =

11.5
SS DNA -0.026
YAL012W 0.467
YAL034C -0.184

Note If you use a cell array of row names or column names to index into a DataMatrix object, the
names must be unique, even though the row names or column names within the DataMatrix object
are not unique.

Use parenthesis indexing to assign new data to a subset of the elements in dmo2:

dmo2({'SS DNA', 'YAL003W'}, 1:2) = [1.700 -0.030; 0.150 -0.130]

dmo2 =

9.5 11.5
SS DNA 1.7 -0.03
YAL003W 0.15 -0.13
YAL012W 0.175 0.467
YAL026C 0.796 0.384
YAL034C 0.487 -0.184

Use parenthesis indexing to delete a subset of the data in dmo2:

dmo2({'SS DNA', 'YAL003W'}, :) = []

dmo2 =

9.5 11.5
YAL012W 0.175 0.467
YAL026C 0.796 0.384
YAL034C 0.487 -0.184

4-7
4 Microarray Analysis

Dot . Indexing

Note In the following examples, notice that when using dot indexing with DataMatrix objects, you
specify all rows or all columns using a colon within single quotation marks, (':').

Use dot indexing to extract the data from the 11.5 column only of dmo:

timeValues = dmo.(':')('11.5')
timeValues =

-0.0260
-0.1290
0.4670
0.3840
-0.1840

Use dot indexing to assign new data to a subset of the elements in dmo:

dmo.(1:2)(':') = 7
dmo =

0 9.5 11.5 13.5

SS DNA 7 7 7 7
YAL003W 7 7 7 7
YAL012W 0.157 0.175 0.467 -0.379
YAL026C 0.246 0.796 0.384 0.981
YAL034C -0.235 0.487 -0.184 -0.669

Use dot indexing to delete an entire variable from dmo:

dmo.YAL034C = []
dmo =

0 9.5 11.5 13.5

SS DNA 7 7 7 7
YAL003W 7 7 7 7
YAL012W 0.157 0.175 0.467 -0.379
YAL026C 0.246 0.796 0.384 0.981

Use dot indexing to delete two columns from dmo:

dmo.(':')(2:3)=[]

dmo =

0 13.5
SS DNA 7 7
YAL003W 7 7
YAL012W 0.157 -0.379
YAL026C 0.246 0.981

4-8
 Representing Expression Data Values in ExptData Objects

Representing Expression Data Values in ExptData Objects

In this section...
“Overview of ExptData Objects” on page 4-9
“Constructing ExptData Objects” on page 4-9
“Using Properties of an ExptData Object” on page 4-10
“Using Methods of an ExptData Object” on page 4-10
“References” on page 4-11

Overview of ExptData Objects

You can use an ExptData object to store expression values from a microarray experiment. An
ExprData object stores the data values in one or more DataMatrix objects, each having the same row
names (feature names) and column names (sample names). Each element (DataMatrix object) in the
ExptData object has an element name.

The following illustrates a small DataMatrix object containing expression values from three samples
(columns) and seven features (rows):

A B C
100001_at 2.26 20.14 31.66
100002_at 158.86 236.25 206.27
100003_at 68.11 105.45 82.92
100004_at 74.32 96.68 84.87
100005_at 75.05 53.17 57.94
100006_at 80.36 42.89 77.21
100007_at 216.64 191.32 219.48

An ExptData object lets you store, manage, and subset the data values from a microarray experiment.
An ExptData object includes properties and methods that let you access, retrieve, and change data
values from a microarray experiment. These properties and methods are useful to view and analyze
the data. For a list of the properties and methods, see ExptData class.

Constructing ExptData Objects

The mouseExprsData.txt file used in this example contains data from Hovatta et al., 2005.

1 Import the bioma.data package so that the DataMatrix and ExptData constructor functions
are available.

import bioma.data.*
2 Use the DataMatrix constructor function to create a DataMatrix object from the gene
expression data in the mouseExprsData.txt file. This file contains a table of expression values
and metadata (sample and feature names) from a microarray experiment done using the
Affymetrix MGU74Av2 GeneChip array. There are 26 sample names (A through Z), and 500
feature names (probe set names).

dmObj = DataMatrix('File', 'mouseExprsData.txt');

3 Use the ExptData constructor function to create an ExptData object from the DataMatrix object.

EDObj = ExptData(dmObj);

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4 Microarray Analysis

4 Display information about the ExptData object, EDObj.

EDObj

Experiment Data:
500 features, 26 samples
1 elements
Element names: Elmt1

Note For complete information on constructing ExptData objects, see ExptData class.

Using Properties of an ExptData Object

To access properties of an ExptData object, use the following syntax:
objectname.propertyname

For example, to determine the number of elements (DataMatrix objects) in an ExptData object:
EDObj.NElements

ans =

To set properties of an ExptData object, use the following syntax:

objectname.propertyname = propertyvalue

For example, to set the Name property of an ExptData object:

EDObj.Name = 'MyExptDataObject'

Note Property names are case sensitive. For a list and description of all properties of an ExptData
object, see ExptData class.

Using Methods of an ExptData Object

To use methods of an ExptData object, use either of the following syntaxes:
objectname.methodname

or
methodname(objectname)

For example, to retrieve the sample names from an ExptData object:

EDObj.sampleNames

Columns 1 through 9

'A' 'B' 'C' 'D' 'E' 'F' 'G' 'H' 'I' ...

To return the size of an ExptData object:

4-10
 Representing Expression Data Values in ExptData Objects

size(EDObj)

ans =

500 26

Note For a complete list of methods of an ExptData object, see ExptData class.

References

[1] Hovatta, I., Tennant, R S., Helton, R., et al. (2005). Glyoxalase 1 and glutathione reductase 1
regulate anxiety in mice. Nature 438, 662–666.

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4 Microarray Analysis

Representing Sample and Feature Metadata in MetaData

Objects
In this section...
“Overview of MetaData Objects” on page 4-12
“Constructing MetaData Objects” on page 4-13
“Using Properties of a MetaData Object” on page 4-15
“Using Methods of a MetaData Object” on page 4-15

Overview of MetaData Objects

You can store either sample or feature metadata from a microarray gene expression experiment in a
MetaData object. The metadata consists of variable names, for example, related to either samples or
microarray features, along with descriptions and values for the variables.

A MetaData object stores the metadata in two dataset arrays:

• Values dataset array — A dataset array containing the measured value of each variable per
sample or feature. In this dataset array, the columns correspond to variables and rows correspond
to either samples or features. The number and names of the columns in this dataset array must
match the number and names of the rows in the Descriptions dataset array. If this dataset array
contains sample metadata, then the number and names of the rows (samples) must match the
number and names of the columns in the DataMatrix objects in the same ExpressionSet object. If
this dataset array contains feature metadata, then the number and names of the rows (features)
must match the number and names of the rows in the DataMatrix objects in the same
ExpressionSet object.
• Descriptions dataset array — A dataset array containing a list of the variable names and their
descriptions. In this dataset array, each row corresponds to a variable. The row names are the
variable names, and a column, named VariableDescription, contains a description of the
variable. The number and names of the rows in the Descriptions dataset array must match the
number and names of the columns in the Values dataset array.

The following illustrates a dataset array containing the measured value of each variable per sample
or feature:
Gender Age Type Strain Source
A 'Male' 8 'Wild type' '129S6/SvEvTac' 'amygdala'
B 'Male' 8 'Wild type' '129S6/SvEvTac' 'amygdala'
C 'Male' 8 'Wild type' '129S6/SvEvTac' 'amygdala'
D 'Male' 8 'Wild type' 'A/J ' 'amygdala'
E 'Male' 8 'Wild type' 'A/J ' 'amygdala'
F 'Male' 8 'Wild type' 'C57BL/6J ' 'amygdala'

The following illustrates a dataset array containing a list of the variable names and their descriptions:
VariableDescription
id 'Sample identifier'
Gender 'Gender of the mouse in study'
Age 'The number of weeks since mouse birth'
Type 'Genetic characters'
Strain 'The mouse strain'
Source 'The tissue source for RNA collection'

4-12
 Representing Sample and Feature Metadata in MetaData Objects

A MetaData object lets you store, manage, and subset the metadata from a microarray experiment. A
MetaData object includes properties and methods that let you access, retrieve, and change metadata
from a microarray experiment. These properties and methods are useful to view and analyze the
metadata. For a list of the properties and methods, see MetaData class

Constructing MetaData Objects

Constructing a MetaData Object from Two dataset Arrays

1 Import the bioma.data package so that the MetaData constructor function is available.

import bioma.data.*
2 Load some sample data, which includes Fisher’s iris data of 5 measurements on a sample of 150
irises.

load fisheriris
3 Create a dataset array from some of Fisher's iris data. The dataset array will contain 750
measured values, one for each of 150 samples (iris replicates) at five variables (species, SL, SW,
PL, PW). In this dataset array, the rows correspond to samples, and the columns correspond to
variables.

irisValues = dataset({nominal(species),'species'}, ...

{meas, 'SL', 'SW', 'PL', 'PW'});
4 Create another dataset array containing a list of the variable names and their descriptions. This
dataset array will contain five rows, each corresponding to the five variables: species, SL, SW, PL,
and PW. The first column will contain the variable name. The second column will have a column
header of VariableDescription and contain a description of the variable.

% Create 5-by-1 cell array of description text for the variables

varDesc = {'Iris species', 'Sepal Length', 'Sepal Width', ...
'Petal Length', 'Petal Width'}';
% Create the dataset array from the variable descriptions
irisVarDesc = dataset(varDesc, ...
'ObsNames', {'species','SL','SW','PL','PW'}, ...
'VarNames', {'VariableDescription'})

irisVarDesc =

VariableDescription
species 'Iris species'
SL 'Sepal Length'
SW 'Sepal Width'
PL 'Petal Length'
PW 'Petal Width'
5 Create a MetaData object from the two dataset arrays.

MDObj1 = MetaData(irisValues, irisVarDesc);

Constructing a MetaData Object from a Text File

1 Import the bioma.datapackage so that the MetaData constructor function is available.

import bioma.data.*
2 View the mouseSampleData.txt file included with the Bioinformatics Toolbox software.

4-13
4 Microarray Analysis

Note that this text file contains two tables. One table contains 130 measured values, one for each
of 26 samples (A through Z) at five variables (Gender, Age, Type, Strain, and Source). In this
table, the rows correspond to samples, and the columns correspond to variables. The second
table has lines prefaced by the # symbol. It contains five rows, each corresponding to the five
variables: Gender, Age, Type, Strain, and Source. The first column contains the variable name.
The second column has a column header of VariableDescription and contains a description
of the variable.

# id: Sample identifier

# Gender: Gender of the mouse in study
# Age: The number of weeks since mouse birth
# Type: Genetic characters
# Strain: The mouse strain
# Source: The tissue source for RNA collection
ID Gender Age Type Strain Source
A Male 8 Wild type 129S6/SvEvTac amygdala
B Male 8 Wild type 129S6/SvEvTac amygdala
C Male 8 Wild type 129S6/SvEvTac amygdala
D Male 8 Wild type A/J amygdala
E Male 8 Wild type A/J amygdala
F Male 8 Wild type C57BL/6J amygdala
G Male 8 Wild type C57BL/6J amygdala
H Male 8 Wild type 129S6/SvEvTac cingulate cortex
I Male 8 Wild type 129S6/SvEvTac cingulate cortex
J Male 8 Wild type A/J cingulate cortex
K Male 8 Wild type A/J cingulate cortex
L Male 8 Wild type A/J cingulate cortex
M Male 8 Wild type C57BL/6J cingulate cortex
N Male 8 Wild type C57BL/6J cingulate cortex
O Male 8 Wild type 129S6/SvEvTac hippocampus
P Male 8 Wild type 129S6/SvEvTac hippocampus
Q Male 8 Wild type A/J hippocampus
R Male 8 Wild type A/J hippocampus
S Male 8 Wild type C57BL/6J hippocampus
T Male 8 Wild type C57BL/6J4 hippocampus
U Male 8 Wild type 129S6/SvEvTac hypothalamus
V Male 8 Wild type 129S6/SvEvTac hypothalamus
W Male 8 Wild type A/J hypothalamus
X Male 8 Wild type A/J hypothalamus
Y Male 8 Wild type C57BL/6J hypothalamus
Z Male 8 Wild type C57BL/6J hypothalamus
3 Create a MetaData object from the metadata in the mouseSampleData.txt file.
MDObj2 = MetaData('File', 'mouseSampleData.txt', 'VarDescChar', '#')

Sample Names:
A, B, ...,Z (26 total)
Variable Names and Meta Information:

VariableDescription
Gender ' Gender of the mouse in study'
Age ' The number of weeks since mouse birth'
Type ' Genetic characters'
Strain ' The mouse strain'
Source ' The tissue source for RNA collection'

For complete information on constructing MetaData objects, see MetaData class.

4-14
 Representing Sample and Feature Metadata in MetaData Objects

Using Properties of a MetaData Object

To access properties of a MetaData object, use the following syntax:

objectname.propertyname

For example, to determine the number of variables in a MetaData object:

MDObj2.NVariables

ans =

To set properties of a MetaData object, use the following syntax:

objectname.propertyname = propertyvalue

For example, to set the Description property of a MetaData object:

MDObj1.Description = 'This is my MetaData object for my sample metadata'

Note Property names are case sensitive. For a list and description of all properties of a MetaData
object, see MetaData class.

Using Methods of a MetaData Object

To use methods of a MetaData object, use either of the following syntaxes:

objectname.methodname

or

methodname(objectname)

For example, to access the dataset array in a MetaData object that contains the variable values:

MDObj2.variableValues;

To access the dataset array of a MetaData object that contains the variable descriptions:

variableDesc(MDObj2)

ans =

VariableDescription
Gender ' Gender of the mouse in study'
Age ' The number of weeks since mouse birth'
Type ' Genetic characters'
Strain ' The mouse strain'
Source ' The tissue source for RNA collection'

Note For a complete list of methods of a MetaData object, see MetaData class.

4-15
4 Microarray Analysis

Representing Experiment Information in a MIAME Object

In this section...
“Overview of MIAME Objects” on page 4-16
“Constructing MIAME Objects” on page 4-16
“Using Properties of a MIAME Object” on page 4-17
“Using Methods of a MIAME Object” on page 4-18

Overview of MIAME Objects

You can store information about experimental methods and conditions from a microarray gene
expression experiment in a MIAME object. It loosely follows the Minimum Information About a
Microarray Experiment (MIAME) specification. It can include information about:

• Experiment design
• Microarrays used
• Samples used
• Sample preparation and labeling
• Hybridization procedures and parameters
• Normalization controls
• Preprocessing information
• Data processing specifications

A MIAME object includes properties and methods that let you access, retrieve, and change
experiment information related to a microarray experiment. These properties and methods are useful
to view and analyze the information. For a list of the properties and methods, see MIAME class.

Constructing MIAME Objects

For complete information on constructing MIAME objects, see MIAME class.

Constructing a MIAME Object from a GEO Structure

1 Import the bioma.data package so that the MIAME constructor function is available.

import bioma.data.*
2 Use the getgeodata function to return a MATLAB structure containing Gene Expression
Omnibus (GEO) Series data related to accession number GSE4616.

geoStruct = getgeodata('GSE4616')

geoStruct =

Header: [1x1 struct]

Data: [12488x12 bioma.data.DataMatrix]
3 Use the MIAME constructor function to create a MIAME object from the structure.

MIAMEObj1 = MIAME(geoStruct);

4-16
 Representing Experiment Information in a MIAME Object

4 Display information about the MIAME object, MIAMEObj.

MIAMEObj1

MIAMEObj1 =

Experiment Description:
Author name: Mika,,Silvennoinen
Riikka,,Kivelä
Maarit,,Lehti
Anna-Maria,,Touvras
Jyrki,,Komulainen
Veikko,,Vihko
Heikki,,Kainulainen
Laboratory: LIKES - Research Center
Contact information: Mika,,Silvennoinen
URL:
PubMedIDs: 17003243
Abstract: A 90 word abstract is available. Use the Abstract property.
Experiment Design: A 234 word summary is available. Use the ExptDesign property.
Other notes:
[1x80 char]

Constructing a MIAME Object from Properties

1 Import the bioma.data package so that theMIAME constructor function is available.

import bioma.data.*
2 Use the MIAME constructor function to create a MIAME object using individual properties.
MIAMEObj2 = MIAME('investigator', 'Jane Researcher',...
'lab', 'One Bioinformatics Laboratory',...
'contact', '[email protected]',...
'url', 'www.lab.not.exist',...
'title', 'Normal vs. Diseased Experiment',...
'abstract', 'Example of using expression data',...
'other', {'Notes:Created from a text file.'});

3 Display information about the MIAME object, MIAMEObj2.

MIAMEObj2

MIAMEObj2 =

Experiment Description:
Author name: Jane Researcher
Laboratory: One Bioinformatics Laboratory
Contact information: [email protected]
URL: www.lab.not.exist
PubMedIDs:
Abstract: A 4 word abstract is available. Use the Abstract property.
No experiment design summary available.
Other notes:
'Notes:Created from a text file.'

Using Properties of a MIAME Object

To access properties of a MIAME object, use the following syntax:

objectname.propertyname

For example, to retrieve the PubMed identifier of publications related to a MIAME object:

MIAMEObj1.PubMedID

ans =

4-17
4 Microarray Analysis

17003243

To set properties of a MIAME object, use the following syntax:

objectname.propertyname = propertyvalue

For example, to set the Laboratory property of a MIAME object:

MIAMEObj1.Laboratory = 'XYZ Lab'

Note Property names are case sensitive. For a list and description of all properties of a MIAME
object, see MIAME class.

Using Methods of a MIAME Object

To use methods of a MIAME object, use either of the following syntaxes:

objectname.methodname

or

methodname(objectname)

For example, to determine if a MIAME object is empty:

MIAMEObj1.isempty

ans =

Note For a complete list of methods of a MIAME object, see MIAME class.

4-18
 Representing All Data in an ExpressionSet Object

Representing All Data in an ExpressionSet Object

In this section...
“Overview of ExpressionSet Objects” on page 4-19
“Constructing ExpressionSet Objects” on page 4-20
“Using Properties of an ExpressionSet Object” on page 4-21
“Using Methods of an ExpressionSet Object” on page 4-21

Overview of ExpressionSet Objects

You can store all microarray experiment data and information in one object by assembling the
following into an ExpressionSet object:

• One ExptData object containing expression values from a microarray experiment in one or more
DataMatrix objects
• One MetaData object containing sample metadata in two dataset arrays
• One MetaData object containing feature metadata in two dataset arrays
• One MIAME object containing experiment descriptions

The following graphic illustrates a typical ExpressionSet object and its component objects.

4-19
4 Microarray Analysis

Each element (DataMatrix object) in the ExpressionSet object has an element name. Also, there is
always one DataMatrix object whose element name is Expressions.

An ExpressionSet object lets you store, manage, and subset the data from a microarray gene
expression experiment. An ExpressionSet object includes properties and methods that let you access,
retrieve, and change data, metadata, and other information about the microarray experiment. These
properties and methods are useful to view and analyze the data. For a list of the properties and
methods, see ExpressionSet class.

Constructing ExpressionSet Objects

Note The following procedure assumes you have executed the example code in the previous sections:

4-20
 Representing All Data in an ExpressionSet Object

• “Representing Expression Data Values in ExptData Objects” on page 4-9

• “Representing Sample and Feature Metadata in MetaData Objects” on page 4-12
• “Representing Experiment Information in a MIAME Object” on page 4-16

1 Import the bioma package so that the ExpressionSet constructor function is available.

import bioma.*
2 Construct an ExpressionSet object from EDObj, an ExptData object, MDObj2, a MetaData object
containing sample variable information, and MIAMEObj, a MIAME object.

ESObj = ExpressionSet(EDObj, 'SData', MDObj2, 'EInfo', MIAMEObj1);

3 Display information about the ExpressionSet object, ESObj.

ESObj

ExpressionSet
Experiment Data: 500 features, 26 samples
Element names: Expressions
Sample Data:
Sample names: A, B, ...,Z (26 total)
Sample variable names and meta information:
Gender: Gender of the mouse in study
Age: The number of weeks since mouse birth
Type: Genetic characters
Strain: The mouse strain
Source: The tissue source for RNA collection
Feature Data: none
Experiment Information: use 'exptInfo(obj)'

For complete information on constructing ExpressionSet objects, see ExpressionSet class.

Using Properties of an ExpressionSet Object

To access properties of an ExpressionSet object, use the following syntax:

objectname.propertyname

For example, to determine the number of samples in an ExpressionSet object:

ESObj.NSamples

ans =

26

Note Property names are case sensitive. For a list and description of all properties of an
ExpressionSet object, see ExpressionSet class.

Using Methods of an ExpressionSet Object

To use methods of an ExpressionSet object, use either of the following syntaxes:

4-21
4 Microarray Analysis

objectname.methodname

or

methodname(objectname)

For example, to retrieve the sample variable names from an ExpressionSet object:

ESObj.sampleVarNames

ans =

'Gender' 'Age' 'Type' 'Strain' 'Source'

To retrieve the experiment information contained in an ExpressionSet object:

exptInfo(ESObj)

ans =

Experiment description
Author name: Mika,,Silvennoinen
Riikka,,Kivelä
Maarit,,Lehti
Anna-Maria,,Touvras
Jyrki,,Komulainen
Veikko,,Vihko
Heikki,,Kainulainen
Laboratory: XYZ Lab
Contact information: Mika,,Silvennoinen
URL:
PubMedIDs: 17003243
Abstract: A 90 word abstract is available Use the Abstract property.
Experiment Design: A 234 word summary is available Use the ExptDesign property.
Other notes:
[1x80 char]

Note For a complete list of methods of an ExpressionSet object, see ExpressionSet class.

4-22
 Visualizing Microarray Images

Visualizing Microarray Images

In this section...
“Overview of the Mouse Example” on page 4-23
“Exploring the Microarray Data Set” on page 4-23
“Spatial Images of Microarray Data” on page 4-25
“Statistics of the Microarrays” on page 4-33
“Scatter Plots of Microarray Data” on page 4-34

Overview of the Mouse Example

This example looks at the various ways to visualize microarray data. The data comes from a
pharmacological model of Parkinson's disease (PD) using a mouse brain. The microarray data for this
example is from Brown, V.M., Ossadtchi, A., Khan, A.H., Yee, S., Lacan, G., Melega, W.P., Cherry, S.R.,
Leahy, R.M., and Smith, D.J.; "Multiplex three dimensional brain gene expression mapping in a mouse
model of Parkinson's disease"; Genome Research 12(6): 868-884 (2002).

The microarray data used in this example is available in a Web supplement to the paper by Brown et
al. and in the file mouse_a1pd.gpr included with the Bioinformatics Toolbox software.

http://labs.pharmacology.ucla.edu/smithlab/genome_multiplex/

The microarray data is also available on the Gene Expression Omnibus Web site at

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30

The GenePix GPR-formatted file mouse_a1pd.gpr contains the data for one of the microarrays used
in the study. This is data from voxel A1 of the brain of a mouse in which a pharmacological model of
Parkinson's disease (PD) was induced using methamphetamine. The voxel sample was labeled with
Cy3 (green) and the control, RNA from a total (not voxelated) normal mouse brain, was labeled with
Cy5 (red). GPR formatted files provide a large amount of information about the array, including the
mean, median, and standard deviation of the foreground and background intensities of each spot at
the 635 nm wavelength (the red, Cy5 channel) and the 532 nm wavelength (the green, Cy3 channel).

Exploring the Microarray Data Set

This procedure illustrates how to import data from the Web into the MATLAB environment, using data
from a study about gene expression in mouse brains as an example. See “Overview of the Mouse
Example” on page 4-23.

1 Read data from a file into a MATLAB structure. For example, in the MATLAB Command Window,
type

pd = gprread('mouse_a1pd.gpr')

Information about the structure displays in the MATLAB Command Window:

pd =
Header: [1x1 struct]
Data: [9504x38 double]
Blocks: [9504x1 double]

4-23
4 Microarray Analysis

Columns: [9504x1 double]

Rows: [9504x1 double]
Names: {9504x1 cell}
IDs: {9504x1 cell}
ColumnNames: {38x1 cell}
Indices: [132x72 double]
Shape: [1x1 struct]
2 Access the fields of a structure using StructureName.FieldName. For example, you can access
the field ColumnNames of the structure pd by typing
pd.ColumnNames

The column names are shown below.

ans =
'X'
'Y'
'Dia.'
'F635 Median'
'F635 Mean'
'F635 SD'
'B635 Median'
'B635 Mean'
'B635 SD'
'% > B635+1SD'
'% > B635+2SD'
'F635 % Sat.'
'F532 Median'
'F532 Mean'
'F532 SD'
'B532 Median'
'B532 Mean'
'B532 SD'
'% > B532+1SD'
'% > B532+2SD'
'F532 % Sat.'
'Ratio of Medians'
'Ratio of Means'
'Median of Ratios'
'Mean of Ratios'
'Ratios SD'
'Rgn Ratio'
'Rgn R²'
'F Pixels'
'B Pixels'
'Sum of Medians'
'Sum of Means'
'Log Ratio'
'F635 Median - B635'
'F532 Median - B532'
'F635 Mean - B635'
'F532 Mean - B532'
'Flags'
3 Access the names of the genes. For example, to list the first 20 gene names, type
pd.Names(1:20)

A list of the first 20 gene names is displayed:

4-24
 Visualizing Microarray Images

ans =
'AA467053'
'AA388323'
'AA387625'
'AA474342'
'Myo1b'
'AA473123'
'AA387579'
'AA387314'
'AA467571'
''
'Spop'
'AA547022'
'AI508784'
'AA413555'
'AA414733'
''
'Snta1'
'AI414419'
'W14393'
'W10596'

Spatial Images of Microarray Data

This procedure illustrates how to visualize microarray data by plotting image maps. The function
maimage can take a microarray data structure and create a pseudocolor image of the data arranged
in the same order as the spots on the array. In other words, maimage plots a spatial plot of the
microarray.

This procedure uses data from a study of gene expression in mouse brains. For a list of field names in
the MATLAB structure pd, see “Exploring the Microarray Data Set” on page 4-23.

1 Plot the median values for the red channel. For example, to plot data from the field F635
Median, type

figure
maimage(pd,'F635 Median')

The MATLAB software plots an image showing the median pixel values for the foreground of the
red (Cy5) channel.

4-25
4 Microarray Analysis

2 Plot the median values for the green channel. For example, to plot data from the field F532
Median, type

figure
maimage(pd,'F532 Median')

The MATLAB software plots an image showing the median pixel values of the foreground of the
green (Cy3) channel.

4-26
 Visualizing Microarray Images

3 Plot the median values for the red background. The field B635 Median shows the median values
for the background of the red channel.

figure
maimage(pd,'B635 Median')

The MATLAB software plots an image for the background of the red channel. Notice the very
high background levels down the right side of the array.

4-27
4 Microarray Analysis

4 Plot the medial values for the green background. The field B532 Median shows the median
values for the background of the green channel.

figure
maimage(pd,'B532 Median')

The MATLAB software plots an image for the background of the green channel.

4-28
 Visualizing Microarray Images

5 The first array was for the Parkinson's disease model mouse. Now read in the data for the same
brain voxel but for the untreated control mouse. In this case, the voxel sample was labeled with
Cy3 and the control, total brain (not voxelated), was labeled with Cy5.

wt = gprread('mouse_a1wt.gpr')

The MATLAB software creates a structure and displays information about the structure.

wt =
Header: [1x1 struct]
Data: [9504x38 double]
Blocks: [9504x1 double]
Columns: [9504x1 double]
Rows: [9504x1 double]
Names: {9504x1 cell}
IDs: {9504x1 cell}
ColumnNames: {38x1 cell}
Indices: [132x72 double]
Shape: [1x1 struct]
6 Use the function maimage to show pseudocolor images of the foreground and background. You
can use the function subplot to put all the plots onto one figure.

figure
subplot(2,2,1);
maimage(wt,'F635 Median')
subplot(2,2,2);
maimage(wt,'F532 Median')
subplot(2,2,3);
maimage(wt,'B635 Median')
subplot(2,2,4);
maimage(wt,'B532 Median')

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4 Microarray Analysis

The MATLAB software plots the images.

7 If you look at the scale for the background images, you will notice that the background levels are
much higher than those for the PD mouse and there appears to be something nonrandom
affecting the background of the Cy3 channel of this slide. Changing the colormap can sometimes
provide more insight into what is going on in pseudocolor plots. For more control over the color,
try the colormapeditor function.

colormap hot

The MATLAB software plots the images.

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 Visualizing Microarray Images

8 The function maimage is a simple way to quickly create pseudocolor images of microarray data.
However if you want more control over plotting, it is easy to create your own plots using the
function imagesc.

First find the column number for the field of interest.

b532MedCol = find(strcmp(wt.ColumnNames,'B532 Median'))

The MATLAB software displays:

b532MedCol =
16
9 Extract that column from the field Data.

b532Data = wt.Data(:,b532MedCol);
10 Use the field Indices to index into the Data.

figure
subplot(1,2,1);
imagesc(b532Data(wt.Indices))
axis image
colorbar
title('B532 Median')

The MATLAB software plots the image.

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4 Microarray Analysis

11 Bound the intensities of the background plot to give more contrast in the image.

maskedData = b532Data;
maskedData(b532Data<500) = 500;
maskedData(b532Data>2000) = 2000;

subplot(1,2,2);
imagesc(maskedData(wt.Indices))
axis image
colorbar
title('Enhanced B532 Median')

The MATLAB software plots the images.

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 Visualizing Microarray Images

Statistics of the Microarrays

This procedure illustrates how to visualize distributions in microarray data. You can use the function
maboxplot to look at the distribution of data in each of the blocks.

1 In the MATLAB Command Window, type

figure
subplot(2,1,1)
maboxplot(pd,'F532 Median','title','Parkinson''s Disease Model Mouse')
subplot(2,1,2)
maboxplot(pd,'B532 Median','title','Parkinson''s Disease Model Mouse')
figure
subplot(2,1,1)
maboxplot(wt,'F532 Median','title','Untreated Mouse')
subplot(2,1,2)
maboxplot(wt,'B532 Median','title','Untreated Mouse')

The MATLAB software plots the images.

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4 Microarray Analysis

2 Compare the plots.

From the box plots you can clearly see the spatial effects in the background intensities. Blocks
numbers 1, 3, 5, and 7 are on the left side of the arrays, and numbers 2, 4, 6, and 8 are on the
right side. The data must be normalized to remove this spatial bias.

Scatter Plots of Microarray Data

This procedure illustrates how to visualize expression levels in microarray data. There are two
columns in the microarray data structure labeled 'F635 Median - B635' and 'F532 Median -

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 Visualizing Microarray Images

B532'. These columns are the differences between the median foreground and the median
background for the 635 nm channel and 532 nm channel respectively. These give a measure of the
actual expression levels, although since the data must first be normalized to remove spatial bias in
the background, you should be careful about using these values without further normalization.
However, in this example no normalization is performed.

1 Rather than working with data in a larger structure, it is often easier to extract the column
numbers and data into separate variables.

cy5DataCol = find(strcmp(wt.ColumnNames,'F635 Median - B635'))

cy3DataCol = find(strcmp(wt.ColumnNames,'F532 Median - B532'))
cy5Data = pd.Data(:,cy5DataCol);
cy3Data = pd.Data(:,cy3DataCol);

The MATLAB software displays:

cy5DataCol =
34

cy3DataCol =
35
2 A simple way to compare the two channels is with a loglog plot. The function maloglog is used
to do this. Points that are above the diagonal in this plot correspond to genes that have higher
expression levels in the A1 voxel than in the brain as a whole.

figure
maloglog(cy5Data,cy3Data)
xlabel('F635 Median - B635 (Control)');
ylabel('F532 Median - B532 (Voxel A1)');

The MATLAB software displays the following messages and plots the images.

Warning: Zero values are ignored

(Type "warning off Bioinfo:MaloglogZeroValues" to suppress
this warning.)
Warning: Negative values are ignored.
(Type "warning off Bioinfo:MaloglogNegativeValues" to suppress
this warning.)

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4 Microarray Analysis

Notice that this function gives some warnings about negative and zero elements. This is because
some of the values in the 'F635 Median - B635' and 'F532 Median - B532' columns are
zero or even less than zero. Spots where this happened might be bad spots or spots that failed to
hybridize. Points with positive, but very small, differences between foreground and background
should also be considered to be bad spots.
3 Disable the display of warnings by using the warning command. Although warnings can be
distracting, it is good practice to investigate why the warnings occurred rather than simply to
ignore them. There might be some systematic reason why they are bad.

warnState = warning; % First save the current warning

state.
% Now turn off the two warnings.
warning('off','Bioinfo:MaloglogZeroValues');
warning('off','Bioinfo:MaloglogNegativeValues');
figure
maloglog(cy5Data,cy3Data) % Create the loglog plot
warning(warnState); % Reset the warning state.
xlabel('F635 Median - B635 (Control)');
ylabel('F532 Median - B532 (Voxel A1)');

The MATLAB software plots the image.

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 Visualizing Microarray Images

4 An alternative to simply ignoring or disabling the warnings is to remove the bad spots from the
data set. You can do this by finding points where either the red or green channel has values less
than or equal to a threshold value. For example, use a threshold value of 10.

threshold = 10;
badPoints = (cy5Data <= threshold) | (cy3Data <= threshold);

The MATLAB software plots the image.

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4 Microarray Analysis

5 You can then remove these points and redraw the loglog plot.

cy5Data(badPoints) = []; cy3Data(badPoints) = [];

figure
maloglog(cy5Data,cy3Data)
xlabel('F635 Median - B635 (Control)');
ylabel('F532 Median - B532 (Voxel A1)');

The MATLAB software plots the image.

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 Visualizing Microarray Images

This plot shows the distribution of points but does not give any indication about which genes
correspond to which points.
6 Add gene labels to the plot. Because some of the data points have been removed, the
corresponding gene IDs must also be removed from the data set before you can use them. The
simplest way to do that is wt.IDs(~badPoints).

maloglog(cy5Data,cy3Data,'labels',wt.IDs(~badPoints),...
'factorlines',2)
xlabel('F635 Median - B635 (Control)');
ylabel('F532 Median - B532 (Voxel A1)');

The MATLAB software plots the image.

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4 Microarray Analysis

7 Try using the mouse to click some of the outlier points.

You will see the gene ID associated with the point. Most of the outliers are below the y = x line.
In fact, most of the points are below this line. Ideally the points should be evenly distributed on
either side of this line.
8 Normalize the points to evenly distribute them on either side of the line. Use the function
manorm to perform global mean normalization.

normcy5 = mannorm(cy5Data);
normcy3 = manorm(cy3Data);

If you plot the normalized data you will see that the points are more evenly distributed about the
y = x line.

figure
maloglog(normcy5,normcy3,'labels',wt.IDs(~badPoints),...
'factorlines',2)
xlabel('F635 Median - B635 (Control)');
ylabel('F532 Median - B532 (Voxel A1)');

The MATLAB software plots the image.

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 Visualizing Microarray Images

9 The function mairplot is used to create an Intensity vs. Ratio plot for the normalized data. This
function works in the same way as the function maloglog.

figure
mairplot(normcy5,normcy3,'labels',wt.IDs(~badPoints),...
'factorlines',2)

The MATLAB software plots the image.

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4 Microarray Analysis

10 You can click the points in this plot to see the name of the gene associated with the plot.

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 5

Phylogenetic Analysis
5 Phylogenetic Analysis

Using the Phylogenetic Tree App

In this section...
“Overview of the Phylogenetic Tree App” on page 5-2
“Opening the Phylogenetic Tree App” on page 5-2
“File Menu” on page 5-3
“Tools Menu” on page 5-11
“Window Menu” on page 5-17
“Help Menu” on page 5-18

Overview of the Phylogenetic Tree App

The Phylogenetic Tree app allows you to view, edit, format, and explore phylogenetic tree data. With
this app you can prune, reorder, rename branches, and explore distances. You can also open or save
Newick or ClustalW tree formatted files. The following sections give a description of menu commands
and features for creating publishable tree figures.

Opening the Phylogenetic Tree App

This section illustrates how to draw a phylogenetic tree from data in a phytree object or a
previously saved file.

The Phylogenetic Tree app can read data from Newick and ClustalW tree formatted files.

This procedure uses the phylogenetic tree data stored in the file pf00002.tree as an example. The
data was retrieved from the protein family (PFAM) Web database and saved to a file using the
accession number PF00002 and the function gethmmtree.

1 Create a phytree object. For example, to create a phytree object from tree data in the file
pf00002.tree, type

tr = phytreeread('pf00002.tree')

The MATLAB software creates a phytree object.

Phylogenetic tree object with 33 leaves (32 branches)

2 View the phylogenetic tree using the app.

phytreeviewer(tr)

Alternatively, click Phylogenetic Tree on the Apps tab.

5-2
 Using the Phylogenetic Tree App

File Menu
The File menu includes the standard commands for opening and closing a file, and it includes
commands to use phytree object data from the MATLAB Workspace. The File menu commands are
shown below.

5-3
5 Phylogenetic Analysis

New Viewer Command

Use the New Viewer command to open tree data from a file into a second Phylogenetic Tree viewer.

1 From the File menu, select New Viewer.

The Open A Phylogenetic Tree dialog box opens.

2 Choose the source for a tree.

5-4
 Using the Phylogenetic Tree App

• MATLAB Workspace — Select the Import from Workspace options, and then select a
phytree object from the list.
• File — Select the Open phylogenetic tree file option, click the Browse button, select a
directory, select a file with the extension .tree, and then click Open. The toolbox uses the
file extension .tree for Newick-formatted files, but you can use any Newick-formatted file
with any extension.

A second Phylogenetic Tree viewer opens with tree data from the selected file.

Open Command

Use the Open command to read tree data from a Newick-formatted file and display that data in the
app.

1 From the File menu, click Open.

The Select Phylogenetic Tree File dialog box opens.

2 Select a directory, select a Newick-formatted file, and then click Open. The app uses the file
extension .tree for Newick-formatted files, but you can use any Newick-formatted file with any
extension.

The app replaces the current tree data with data from the selected file.

Import from Workspace Command

Use the Import from Workspace command to read tree data from a phytree object in the MATLAB
Workspace and display the data using the app.

1 From the File menu, select Import from Workspace.

The Get Phytree Object dialog box opens.

5-5
5 Phylogenetic Analysis

2 From the list, select a phytree object in the MATLAB Workspace.

3 Click the Import button.

The app replaces the current tree data with data from the selected object.

Open Original in New Viewer

There may be times when you make changes that you would like to undo. The Phylogenetic Tree
app does not have an undo command, but you can get back to the original tree you started viewing
with the Open Original in New Viewer command.

From the File menu, select Open Original in New Viewer.

A new Phylogenetic Tree viewer opens with the original tree.

Save As Command

After you create a phytree object or prune a tree from existing data, you can save the resulting tree
in a Newick-formatted file. The sequence data used to create the phytree object is not saved with
the tree.

1 From the File menu, select Save As.

The Save Phylogenetic tree as dialog box opens.

2 In the Filename box, enter the name of a file. The toolbox uses the file extension .tree for
Newick-formatted files, but you can use any file extension.
3 Click Save.

The app saves tree data without the deleted branches, and it saves changes to branch and leaf
names. Formatting changes such as branch rotations, collapsed branches, and zoom settings are
not saved in the file.

Export to New Viewer Command

Because some of the Phylogenetic Tree viewer commands cannot be undone (for example, the Prune
command), you might want to make a copy of your tree before trying a command. At other times, you

5-6
 Using the Phylogenetic Tree App

might want to compare two views of the same tree, and copying a tree to a new tool window allows
you to make changes to both tree views independently .

1 Select File > Export to New Viewer, and then select either With Hidden Nodes or Only
Displayed.

A new Phylogenetic Tree viewer opens with a copy of the tree.

2 Use the new figure to continue your analysis.

Export to Workspace Command

The Phylogenetic Tree app can open Newick-formatted files with tree data. However, it does not
create a phytree object in the MATLAB Workspace. If you want to programmatically explore
phylogenetic trees, you need to use the Export to Workspace command.

1 Select File > Export to Workspace, and then select either With Hidden Nodes or Only
Displayed.

The Export to Workspace dialog box opens.

2 In the Workspace variable name box, enter the name for your phylogenetic tree data. For
example, enter MyTree.

3 Click OK.

The app creates a phytree object in the MATLAB Workspace.

Print to Figure Command

After you have explored the relationships between branches and leaves in your tree, you can copy the
tree to a MATLAB Figure window. Using a Figure window lets you use all the features for annotating,
changing font characteristics, and getting your figure ready for publication. Also, from the Figure
window, you can save an image of the tree as it was displayed in the Phylogenetic Tree app.

1 From the File menu, select Print to Figure, and then select either With Hidden Nodes or
Only Displayed.

The Print Phylogenetic Tree to Figure dialog box opens.

5-7
5 Phylogenetic Analysis

2 Select one of the Rendering Types.

Rendering Type Description

'square' (default)

5-8
 Using the Phylogenetic Tree App

Rendering Type Description

'angular'

'radial'

5-9
5 Phylogenetic Analysis

Rendering Type Description

'equalangle'

Tip This rendering type hides the significance of the root node
and emphasizes clusters, thereby making it useful for visually
assessing clusters and detecting outliers.
'equaldaylight'

Tip This rendering type hides the significance of the root node
and emphasizes clusters, thereby making it useful for visually
assessing clusters and detecting outliers.
3 Select the Display Labels you want on your figure. You can select from all to none of the
options.

• Branch Nodes — Display branch node names on the figure.

• Leaf Nodes — Display leaf node names on the figure.
• Terminal Nodes — Display terminal node names on the right border.
4 Click the Print button.

A new Figure window opens with the characteristics you selected.

Print Preview Command

When you print from the Phylogenetic Tree app or a MATLAB Figure window (with a tree published
from the viewer), you can specify setup options for printing a tree.

5-10
 Using the Phylogenetic Tree App

1 From the File menu, select Print Preview.

The Print Preview window opens, which you can use to select page formatting options.

2 Select the page formatting options and values you want, and then click Print.

Print Command

Use the Print command to make a copy of your phylogenetic tree after you use the Print Preview
command to select formatting options.

1 From the File menu, select Print.

The Print dialog box opens.

2 From the Name list, select a printer, and then click OK.

Tools Menu
Use the Tools menu to:

• Explore branch paths

• Rotate branches
• Find, rename, hide, and prune branches and leaves.

The Tools menu and toolbar contain most of the commands specific to trees and phylogenetic
analysis. Use these commands and modes to edit and format your tree interactively. The Tools menu
commands are:

5-11
5 Phylogenetic Analysis

Inspect Mode

Viewing a phylogenetic tree in the Phylogenetic Tree app provides a rough idea of how closely
related two sequences are. However, to see exactly how closely related two sequences are, measure
the distance of the path between them. Use the Inspect command to display and measure the path
between two sequences.

1
Select Tools > Inspect, or from the toolbar, click the Inspect Tool Mode icon .

The app is set to inspect mode.

2 Click a branch or leaf node (selected node), and then hover your cursor over another branch or
leaf node (current node).

The app highlights the path between the two nodes and displays the path length in the pop-up
window. The path length is the patristic distance calculated by the seqpdist function.

5-12
 Using the Phylogenetic Tree App

Collapse and Expand Branch Mode

Some trees have thousands of leaf and branch nodes. Displaying all the nodes can create an
unreadable tree diagram. By collapsing some branches, you can better see the relationships between
the remaining nodes.
1 Select Tools > Collapse/Expand, or from the toolbar, click the Collapse/Expand Brand Mode

icon .

The app is set to collapse/expand mode.

2 Point to a branch.

The paths, branch nodes, and leaf nodes below the selected branch appear in gray, indicating you
selected them to collapse (hide from view).

3 Click the branch node.

The app hides the display of paths, branch nodes, and leaf nodes below the selected branch.
However, it does not remove the data.

4 To expand a collapsed branch, click it or select Tools > Reset View.

Tip After collapsing nodes, you can redraw the tree by selecting Tools > Fit to Window.

Rotate Branch Mode

A phylogenetic tree is initially created by pairing the two most similar sequences and then adding the
remaining sequences in a decreasing order of similarity. You can rotate branches to emphasize the
direction of evolution.
1
Select Tools > Rotate Branch, or from the toolbar, click the Rotate Branch Mode icon .

The app is set to rotate branch mode.

2 Point to a branch node.

5-13
5 Phylogenetic Analysis

3 Click the branch node.

The branch and leaf nodes below the selected branch node rotate 180 degrees around the branch
node.
4 To undo the rotation, simply click the branch node again.

Rename Leaf or Branch Mode

The Phylogenetic Tree app takes the node names from a phytree object and creates numbered
branch names starting with Branch 1. You can edit any of the leaf or branch names.

1
Select Tools > Rename, or from the toolbar, click the Rename Leaf/Branch Mode icon .

The app is set to rename mode.

2 Click a branch or leaf node.

A text box opens with the current name of the node.

3 In the text box, edit or enter a new name.

4 To accept your changes and close the text box, click outside of the text box. To save your
changes, select File > Save As.

Prune (Delete) Leaf or Branch Mode

Your tree can contain leaves that are far outside the phylogeny, or it can have duplicate leaves that
you want to remove.

1 Select Tools > Prune, or from the toolbar, click the Prune (delete) Leaf/Branch Mode icon

The app is set to prune mode.

2 Point to a branch or leaf node.

5-14
 Using the Phylogenetic Tree App

For a leaf node, the branch line connected to the leaf appears in gray. For a branch node, the
branch lines below the node appear in gray.

Note If you delete nodes (branches or leaves), you cannot undo the changes. The Phylogenetic
Tree app does not have an Undo command.
3 Click the branch or leaf node.

The tool removes the branch from the figure and rearranges the other nodes to balance the tree
structure. It does not recalculate the phylogeny.

Tip After pruning nodes, you can redraw the tree by selecting Tools > Fit to Window.

Zoom In, Zoom Out, and Pan Commands

The Zoom and Pan commands are the standard controls for resizing and moving the screen in any
MATLAB Figure window.

1
Select Tools > Zoom In, or from the toolbar, click the Zoom In icon .

The app activates zoom in mode and changes the cursor to a magnifying glass.

2 Place the cursor over the section of the tree diagram you want to enlarge and then click.

The tree diagram doubles its size.

3
From the toolbar click the Pan icon .

5-15
5 Phylogenetic Analysis

4 Move the cursor over the tree diagram, left-click, and drag the diagram to the location you want
to view.

Tip After zooming and panning, you can reset the tree to its original view, by selecting Tools >
Reset View.

Select Submenu

Select a single branch or leaf node by clicking it. Select multiple branch or leaf nodes by Shift-
clicking the nodes, or click-dragging to draw a box around nodes.

Use the Select submenu to select specific branch and leaf nodes based on different criteria.

• Select By Distance — Displays a slider bar at the top of the window, which you slide to specify a
distance threshold. Nodes whose distance from the selected node are below this threshold appear
in red. Nodes whose distance from the selected node are above this threshold appear in blue.
• Select Common Ancestor — For all selected nodes, highlights the closest common ancestor
branch node in red.
• Select Leaves — If one or more nodes are selected, highlights the nodes that are leaf nodes in
red. If no nodes are selected, highlights all leaf nodes in red
• Propagate Selection — For all selected nodes, highlights the descendant nodes in red.
• Swap Selection — Clears all selected nodes and selects all deselected nodes.

After selecting nodes using one of the previous commands, hide and show the nodes using the
following commands:

• Collapse Selected
• Expand Selected
• Expand All

Clear all selected nodes by clicking anywhere else in the Phylogenetic Tree app.

Find Leaf or Branch Command

Phylogenetic trees can have thousands of leaves and branches, and finding a specific node can be
difficult. Use the Find Leaf/Branch command to locate a node using its name or part of its name.

1 Select Tools > Find Leaf/Branch.

The Find Leaf/Branch dialog box opens.

5-16
 Using the Phylogenetic Tree App

2 In the Regular Expression to match box, enter a name or partial name of a branch or leaf
node.
3 Click OK.

The branch or leaf nodes that match the expression appear in red.

After selecting nodes using the Find Leaf/Branch command, you can hide and show the nodes using
the following commands:

• Collapse Selected
• Expand Selected
• Expand All

Collapse Selected, Expand Selected, and Expand All Commands

When you select nodes, either manually or using the previous commands, you can then collapse them
by selecting Tools > Collapse Selected.

The data for branches and leaves that you hide using the Collapse/Expand or Collapse Selected
command are not removed from the tree. You can display selected or all hidden data using the
Expand Selected or Expand All command.

Fit to Window Command

After you hide nodes with the collapse commands, or delete nodes with the Prune command, there
can be extra space in the tree diagram. Use the Fit to Window command to redraw the tree diagram
to fill the entire Figure window.

Select Tools > Fit to Window.

Reset View Command

Use the Reset View command to remove formatting changes such as collapsed branches and zooms.

Select Tools > Reset View.

Options Submenu

Use the Options command to select the behavior for the zoom and pan modes.

• Unconstrained Zoom — Allow zooming in both horizontal and vertical directions.

• Horizontal Zoom — Restrict zooming to the horizontal direction.
• Vertical Zoom (default) — Restrict zooming to the vertical direction.
• Unconstrained Pan — Allow panning in both horizontal and vertical directions.
• Horizontal Pan — Restrict panning to the horizontal direction.
• Vertical Pan (default) — Restrict panning to the vertical direction.

Window Menu
This section illustrates how to switch to any open window.

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5 Phylogenetic Analysis

The Window menu is standard on MATLAB interfaces and Figure windows. Use this menu to select
any opened window.

Help Menu
This section illustrates how to select quick links to the Bioinformatics Toolbox documentation for
phylogenetic analysis functions, tutorials, and the Phylogenetic Tree app reference.

Use the Help menu to select quick links to the Bioinformatics Toolbox documentation for
phylogenetic analysis functions, tutorials, and the phytreeviewer reference.

5-18