Introduction to

next-generation sequencing
Ion Torrent technology and instrumentation
Introduction
DNA sequencing involves determining the order of nucleic acid residues in biological
samples, and it is key to understanding the way that genomes function. Sequencing
information forms the basis of much of modern biological inquiry, and it is integral to a great
variety of research and clinical applications [1]. The techniques and technology of DNA and
RNA sequencing have been developed over more than 50 years, and many of the technical
improvements available today were instigated by the Human Genome Project [2]. Since
completion of that project, the speed of sequencing has increased even as the costs have
decreased, making the most current technological advances available to a wider range of
laboratories than ever before.

Sequencing is an important component of biological research because it informs so many

areas of biology, medicine, epidemiology, and forensics. Just a few of the applications of
sequencing include the determination of the roles of genes and gene regulation in infectious
and inherited diseases and in cancers, the analysis of phylogenetic relationships of various
organisms, and forensic examination of highly complex biological samples. One of the most
exciting developments has been the invention of next-generation sequencing, or NGS. NGS
was first introduced for commercial use in 2005, and it would not be an overstatement to
say that it has revolutionized the various fields that utilize genetic analysis [3]. NGS allows for
the interrogation of up to thousands of genes at a single time from multiple samples and the
discovery and analysis of a broad variety of features of the genome. It has the advantages
of requiring low amounts of sample input, high accuracy, and the ability to detect low-
frequency variants [3].

Thermo Fisher Scientific has developed a wide array of Ion Torrent™ products for NGS,
including instrumentation, assay kits, reagents, and consumables. This ebook describes
the different types and methods of NGS that are currently available, and the applications to
which they are relevant. In particular, we highlight the use of Ion Torrent products to address
a broad range of questions that may be answerable with NGS data.

References
1. Heather JM, Chain B (2016) The sequence of sequencers: the history of sequencing DNA. Genomics 107:1–8. doi: 10.1016/j.ygeno.2015.11.003.
2. National Human Genome Research Institute (2020) DNA sequencing fact sheet. https://www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Fact-Sheet.
3. Thermo Fisher Scientific (n.d.) What is next-generation sequencing (NGS)? https://www.thermofisher.com/us/en/home/life-science/sequencing/sequencing-learning-center/
next-generation-sequencing-information/ngs-basics/what-is-next-generation-sequencing.html.
Contents

Chapter 1: Why NGS matters ..........................................................................................................................................4

Overview........................................................................................................................................................................... 4
Genomic aberrations detected by NGS............................................................................................................................. 4
Advantages of NGS........................................................................................................................................................... 4
Next-generation sequencing methods............................................................................................................................... 5
Whole-genome sequencing................................................................................................................................6
Targeted sequencing..........................................................................................................................................6
Three advantages of amplicon-based approaches..............................................................................................7
The NGS workflows.......................................................................................................................................................... 8
How to select the optimal NGS method............................................................................................................................. 8
Conclusion........................................................................................................................................................................ 9

Chapter 2: Introduction to Ion Torrent sequencing..........................................................................................................10

Overview......................................................................................................................................................................... 10
How Ion Torrent sequencing works................................................................................................................................. 10
How the technology is used to call a base....................................................................................................................... 10

Chapter 3: NGS instruments—the Ion GeneStudio S5 series.......................................................................................... 12

Overview......................................................................................................................................................................... 12
Key features of Ion GeneStudio S5 systems.................................................................................................................... 12
Application highlights of the Ion GeneStudio S5 systems................................................................................................ 13
Cancer research............................................................................................................................................... 13
Complex and inherited disease research........................................................................................................... 14
Reproductive health research............................................................................................................................ 14
Microbiology and infectious disease research................................................................................................... 14
Conclusion...................................................................................................................................................................... 15

Chapter 4: NGS instruments—the Genexus System.......................................................................................................16

Overview......................................................................................................................................................................... 16
The Genexus Purification System.................................................................................................................................... 16
The Genexus Integrated Sequencer.................................................................................................................................17
Application highlights of the Genexus System .................................................................................................................17
Oncology clinical research solutions.................................................................................................................. 17
Microbial and infectious disease research......................................................................................................... 18
Coronavirus (SARS-CoV-2) research................................................................................................................. 18
Inherited disease clinical research solutions......................................................................................................19
Conclusion..................................................................................................................................................................... 21
Chapter 1: Why NGS matters

Overview • Constitutional copy number variants (CNVs) are DNA segments

The speed, throughput, and accuracy of next-generation present at a variable copy number in comparison to the
number in a normal genome. CNVs lead to gene amplification
sequencing (NGS) have revolutionized genetic analysis and
and deletion, which can disrupt protein production, biological
enabled new applications in genomic and clinical research,
pathways, and cell function; they can potentially lead to
precision medicine, reproductive health, and beyond. disease development.

NGS is a technology for determining the sequence of DNA or • Insertions and deletions (indels) are additions or deletions of
RNA to study genetic variation associated with diseases or one or more nucleotides in a DNA sequence. They are the
other biological phenomena. NGS was initially introduced for second most common aberrations in the human genome (after
SNVs) and are well known to contribute to disease.
commercial use in 2005. It was known then as “massively parallel
sequencing” because it enabled the sequencing of many DNA • Single-nucleotide variants (SNVs) are single-base changes
strands simultaneously, instead of one at a time as with traditional in DNA sequences responsible for genetic diversity that also
Sanger sequencing by capillary electrophoresis (CE). Both NGS may contribute to the development of complex diseases, such
as cancer.
and Sanger sequencing have utility in today’s genetic analysis
environment. Sanger sequencing is best for analyzing small
Advantages of NGS
numbers of gene targets and samples and can be accomplished
• Time savings: A comprehensive molecular profile can be
in a single day. NGS results are often verified using Sanger
available in as little as 24 hours, accelerating research that has
sequencing because the latter is considered the gold-standard the power to advance science.
sequencing technology.
• Tissue savings: NGS can provide comprehensive genomic
NGS enables the interrogation of hundreds to thousands of profile results from one small sample with as little as 10 ng of
genes at one time in multiple samples. It also allows for the DNA or RNA.
discovery and analysis of different types of genomic features in a • Cost and resource savings: If three or more biomarkers are
single sequencing run, from single-nucleotide variants (SNVs) to required, it can be cheaper to run one NGS test than multiple
copy number and structural variants, and even RNA fusions. NGS single-gene assays. NGS also allows savings by streamlining
allows ideal throughput per run, and studies can be performed training and technical experience requirements, as well as
instrumentation maintenance, on one platform. Additionally, the
quickly and cost-effectively. Additional advantages of NGS
use of a single platform saves on space requirements.
include lower sample input requirements and the ability to detect
variants at lower allele frequencies than with Sanger sequencing. • High specificity and sensitivity: NGS enables detection of
genomic variants even if they are present in extremely low
Genomic aberrations detected fractions of cells in the sample (such as in liquid biopsies) and
allows researchers to distinguish between different cells.
by NGS
• Reduced sample handling errors: The more tests that are
NGS can be used for many biomarkers and biomarker types (e.g.,
performed to achieve the required result, the more potential
mutations, fusions, and copy number variations) simultaneously there is for errors to occur. Consolidation into one NGS test
with a single test. can reduce error rates.

• Translocations are migrations of DNA from one chromosome

to another. They lead to the origination of new fusion genes
and proteins and play a significant role in disease development.
Only those translocations that are transcribed to RNA can
potentially become functional, and therefore RNA sequencing
is the preferred method for fusion testing.

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Next-generation sequencing methods
Genomics researchers have multiple NGS methods to choose While whole-genome and whole-exome sequencing are suited
from when designing and implementing their studies. General to discovery-based questions, targeted NGS is preferred for the
NGS methods include whole-genome sequencing (WGS) and study of known variants or rare alleles. Additionally, within each
targeted sequencing, which is further subdivided into exome of these approaches, there are specialized techniques tailored
sequencing and gene- or region-specific panels (Table 1.1). to specific sample types, organisms, diseases, or regions of
Although all these methods can yield gene sequences of interest the genome. Table 1.1 summarizes the pros and cons of each
to a researcher, they are far from interchangeable. method and includes examples and applications.

Table 1.1. Comparison of DNA sequencing methods.

Whole-genome sequencing Targeted sequencing: exome Targeted sequencing: gene-
(WGS) sequencing or region-specific panels
Description Sequencing of the entire Sequencing of only exons Sequencing of regions of
genome (protein-coding regions) interest such as disease-
associated genes or
genomic hotspots
Pros • Most comprehensive • Exomes constitute 1% of the • Highly flexible,
genome coverage human genome—much less customizable designs
data to analyze than for WGS
• Detects the widest range • Data are focused specifically
of features: SNVs, indels, • Faster workflow than that of on regions or genes of
structural and copy WGS interest
number variants, and
• Used to multiplex a small • Lowest sample input (10 ng)
regulatory elements
number of samples
• Compatible with FFPE tissue
• No bias from PCR
• Medium sample input samples
amplification or probe (50 ng–1 μg depending on
hybridization • Used to multiplex large
library prep method)
numbers of samples
• Best for discovery research
• Better for detecting rare
alleles
Cons • Great amount of potentially • Only sequences data in • Only obtains data on
unnecessary data exons (may miss functionally targeted regions (may miss
from noncoding and relevant variants) relevant variants if not in the
nonfunctional regions design)
• May generate too much
• Data are very complicated extra data if one only needs
to study a small number of
• Multiplexing is usually not genes
possible
Speed of results Slow Medium Fast
Cost $$$ $$ $
Data volume Large Medium Small
When to use • Complete coverage of • Disease-specific • Clinical research
genome needed research projects sample sequencing
• De novo assembly • Clinical research • Disease-specific
sample sequencing research projects
• Discovery of unknown
genomic variants associated • In vitro diagnostic
with a disease (IVD) testing
• Aneuploidy detection • Creation of laboratory-
(preimplantation genetic developed tests (LDT)
testing)
• Inherited disease detection
and characterization
• Oncology applications
• Immune repertoire analysis
• Liquid biopsy samples

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Whole-genome sequencing By sequencing only what you need, cost benefits are realized
WGS is the most comprehensive sequencing method that from less intensive computational processing, informatics, and
enables an in-depth analysis of entire genomes, including exons, data storage; shorter workflows and sequencing time; and
noncoding regions, and structural variants. WGS libraries are higher depth of coverage for rare variants that occur at low allelic
typically prepared using fragmentation or enzymatic digestion frequency. More samples can be processed simultaneously in a
of genomic DNA. WGS does not require prior knowledge single sequencing run for a faster time-to-result, whether for an
of the genome sequence being analyzed, so it is the best individual sample or a cohort. Whole-exome sequencing (WES)
method for discovery of new genetic variations associated with and target-specific panels are the two most common targeted
disease, de novo genome assembly, microbial sequencing, sequencing approaches.
and low-pass genome sequencing for copy number and
• WES enables analysis of all the exons (the protein-coding
aneuploidy determination.
regions of the genome). About 20,000 genes constitute the
While WGS has advanced discovery and human health, certain human exome, which accounts for approximately 1% of the
entire genome. WES allows researchers to focus on content
complex regions of the genome are difficult to analyze with this
that may be more relevant to disease compared to the whole
approach. It should be pointed out here that WGS in humans has genome sequence.
resulted in a population sequencing bias, with existing databases
• Target-specific NGS panels are the most flexible option
noted to be neither complete nor accurate. For many research
because they can be designed to sequence any gene or
applications, the cost of computational processing, informatics,
region of interest in a genome and can include structural and
and data storage needs for whole-genome analysis is a burden. copy number variation, as well as RNA transcript analysis.
The extra cost is of little benefit when studying a specific region Unlike broader approaches such as WES or WGS, targeted
of interest associated with a disease or in translational research panels generate smaller and more manageable data sets,
applications. To address this issue, many researchers use which reduces the data analysis burden for researchers.
Using target-specific panels is the fastest and most
targeted sequencing approaches, such as exome sequencing
cost-effective NGS method, making them more suitable for
or smaller gene- or region-specific panels to improve sequence
clinical research applications.
coverage and reduce their total sequencing workflow costs.
• Targeted NGS libraries enriched for specific genomic regions
can be made using two techniques: hybridization capture or
Targeted sequencing
amplicon-based enrichment.
A targeted NGS approach leverages current knowledge of the
genome and uses molecular biology methods to enrich it for
specific sequences. The targeted approach enables researchers
to focus their studies on the individual genes or genomic regions
that are most relevant to their research. Using this targeted
method, obtaining sequence coverage of challenging genomic
regions is now possible, including for regions from difficult-to-
sequence or limited samples, such as degraded DNA or RNA
from clinical samples, or circulating DNA from blood samples.

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Hybridization capture uses molecules complementary to target 2. Amplicon-based enrichment can be used to
regions to act as probes that select the target molecules from the discriminate between two highly homologous regions
sample. These capture probes are either immobilized on a solid in the genome because PCR primers can be designed
substrate in an array-based format or used directly in solution. to specifically target the desired region.
In the array-based format, the DNA sample is applied to the In contrast, hybridization capture would have difficulty
solid surface and the targeted DNA fragments hybridize to the distinguishing between the two homologous regions in the
immobilized capture probes. Any unbound molecules are washed genome, resulting in nonspecific enrichment. For example, the
away, leaving the desired targets on the surface. The desired PTEN gene is a known tumor suppressor gene that controls
isolated genetic material is now eluted away and amplified for cell growth and division. PTEN is one of the most commonly
sequencing. With solution-based hybridization, the probes are mutated suppressor genes in cancer. PTENP1 is a processed
biotinylated and hybridized targets are isolated and purified for pseudogene very similar in sequence, with a missense mutation
subsequent amplification using streptavidin magnetic beads. in the initiation methionine codon that prevents translation of the
normal PTEN protein. The ability to distinguish and target the
Amplicon-based enrichment uses highly multiplex PCR that is
correct gene clearly plays an important role in cancer research.
carefully designed to amplify regions of interest from the DNA or
The same concept applies when trying to target low-complexity
complementary DNA (cDNA) sample (Figure 1.1). This workflow
regions that are prevalent in whole genomes, such as
is much shorter than the hybridization capture workflow. After
dinucleotide and trinucleotide repeats.
multiplex PCR, the resulting amplicon library is purified from
the sample material, ligated to sequencing adaptors containing 3. Amplicon-based enrichment can be used to better
barcodes, and used for sequencing. Amplicon-based enrichment detect known insertions and fusion events than
techniques have several advantages compared to hybridization capture hybridization.
capture methods: Since capture hybridization requires developing complementary
capture probes against a known reference genome, unknown
Three advantages of amplicon-based approaches genetic mutations could disrupt the hybridization process
1. PCR specificity allows researchers to enrich for and result in a failure to enrich for a target region of interest.
target gene regions from low sample input amounts. This issue is particularly relevant for genetic regions that
Limited sample sources with trace amounts of DNA and/or RNA, have many variants near one another. Hypervariable regions,
such as FFPE tissue, fine needle aspirates, or circulating tumor such as the T cell immune repertoire, can be sequenced
DNA (ctDNA), can now be sequenced for biomarker discovery more effectively using amplicon-based enrichment, providing
and retrospective clinical trials. translational researchers a tool to discover predictive biomarkers
Amplicon-based next-generation forsequencing
immunotherapy.
priciple

1 DNA and RNA are extracted

from an FFPE sample 5
Hydrogen ions are released, changing
the solution pH, as complementary
nucleotides are added

Regions of interest are pH changes are measured,

2 amplified from fragmented DNA
and barcode labeled
6 converted to voltage, and then
used to determine the base call
G A T C

Each labeled fragment is Integrated software assembles

3 attached to its own bead and
copied, until it covers the bead 7 and analyzes the data to identify
target gene variants, if present in
the sequence

4 Prepared DNA fragments, called

amplicons, are sequenced in a
massively parallel fashion
Figure 1.1. Workflow for amplicon-based NGS.

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 How to select the optimal
The NGS workflows NGS method
Prepare the sample and library While NGS is the ideal platform for testing multiple biomarkers
Begin the NGS workflow by preparing a sequencing library from and preserving precious sample tissue, not all NGS is the same.
the tissue sample. First, extract and purify the DNA and/or RNA, These are the six key factors to consider when choosing the NGS
depending on the assay. Using PCR, amplify regions of interest to technology for your laboratory:
generate an amplicon sequencing library. You can either prepare
1. Adequate portfolio of applications and panel coverage—
a library from a single specimen or combine multiple specimens
In precision medicine research, one size does not fit all. You need
into a mixed library. In a process called sample multiplexing, each
a panel that includes genes relevant to the disease of interest
specimen is tagged with a specific barcode so it can be analyzed
and that can profile your sample format. For example, if you
independently downstream. Multiplexing enables you to optimize
are testing non-small cell lung cancer (NSCLC) samples, you
efficiency by maximizing the number of samples processed in
need a panel that includes ALK, ROS1, EGFR, BRAF, NTRK, and
each sequencing run.
RET, and likely also offers the possibility to profile liquid biopsy
samples, as in many cases there is not enough tissue available.
Create the template
For immuno-oncology clinical research, both tumor mutational
After the sequencing library is prepared, generate a template in
burden (TMB) sequencing and T cell receptor (TCR) sequencing
preparation for sequencing. This entire process can be easily
are becoming important.
automated. During template generation, the library is settled
onto a solid substrate and further amplified. For Ion Torrent™ 2. Panel design—Depending on the variant to be detected,
technology, the substrate is a semi-conductor microchip that either DNA- or RNA-based sequencing is preferable. Often,
enables the sequence of each amplicon in the library to be an assay that can do both at once is required. RNA-based
read independently. methodology is optimal for testing fusions, as it directly detects
translocation events between the target gene and partner gene. It
Sequence should also be able to detect not only all the known, but also the
Once the template is ready, simply load the chip into the novel, driver and partner combinations.
sequencer and initiate a sequencing run. The sequence of
3. Sample requirements—Often the amount of tissue that is
each amplicon in the library will be read during this process,
available is very limited. Different NGS methods vary significantly
and the data will be digitally transmitted to a computer for
in the amount of sample required, ranging from 10 to 500 ng of
downstream analysis.
nucleic acid (RNA or DNA), which can have a direct impact on
your ability to successfully test all samples.
Analyze data and interpret results
Integrated analysis software assembles the amplicon sequencing 4. Completeness and automation level of the workflow—NGS
data and calls any identified variants. Using a decision support workflows can be complex. An easy-to-use and highly automated
tool, gene variants detected in the sequences can be matched workflow from sample to report simplifies laboratory operation
against databases of known relevant biomarkers, associated and test implementation.
therapies, clinical trials, and guidelines.
5. Analytical validation support—High-touch consultation
service and support from the vendor help accelerate a
laboratory’s validation process to implement the test in a
time-efficient manner and save costs.

6. Robustness and key performance characteristics—Not

all NGS technologies can handle all tissue types equally well.
For example, FFPE material can be challenging, especially
when there is insufficient tumor sample. Look for evidence of
the sequencing success rate, or the opposite—failure rate and
“quantity not sufficient” frequency. Specificity and sensitivity
are very important, as cell populations may be heterogeneous,
and a particular aberration may be present only in a small
proportion of cells, although the patient might still benefit from
the corresponding therapy. High reproducibility is also required,
so you can generate consistent results.
8 Contents
Conclusion
There are many possible research applications for NGS. These include WGS to identify
associations of genomic variation with different diseases as well as targeted NGS, in
which the whole power of NGS is used to interrogate a defined number of targets with
possible significance in disease pathogenesis.

9 Contents
Chapter 2: Introduction to
Ion Torrent sequencing
Overview
Ion Torrent™ technology from Thermo Fisher Scientific takes a
unique approach to next-generation sequencing (NGS). This
approach marries simple chemistry to proprietary semiconductor
technology, providing a fast, simple, affordable, and scalable
sequencing solution.

How Ion Torrent sequencing works How the technology is used to call
Ion Torrent technology directly translates chemically encoded a base
information (A, C, G, or T) into digital information (0 or 1) on a
In nature, when a nucleotide is incorporated into a strand of DNA
semiconductor chip.
by a polymerase, a hydrogen ion is released as a byproduct. This
The sequencing process starts when a sample of DNA is cut is how an Ion Torrent system sequences DNA.
into millions of fragments. Each fragment is then attached
If a nucleotide is added to a DNA template and is then
to its own bead and is copied until it covers the bead. This
incorporated into a strand of DNA, a hydrogen ion will be
automated process covers millions of beads with millions of
released. The charge from that ion will change the pH of the
different DNA fragments. These beads then flow across the
solution in the well. Our proprietary ion sensors beneath the well
chip, each depositing into a well (Figure 2.1). The Ion Torrent™
measure the change in pH and convert it to voltage. In essence,
next-generation sequencer then sequentially floods the chip with
each well works as the world’s smallest pH meter.
nucleotides to initiate base calling.

Figure 2.1. Ion Torrent technology. Beads covered with DNA fragments flowing across the chip and depositing into a well.

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The sequencer records the voltage, indicating the nucleotide The Ion Torrent™ chips help you scale the workflow to your
was incorporated and the base was called, going directly from research needs so that you can run both small- and large-
chemical information to digital information. For example, if a scale projects without the need to change platforms. The
polymerase incorporates a C nucleotide in a DNA strand when a semiconductor approach helps you implement a significantly
complementary G molecule is present, a hydrogen ion is released faster NGS workflow compared with other sequencing
and there is a voltage change (Figure 2.2A). If the next nucleotide technologies. Ion Torrent technology harnesses the power of
that floods the chip is not a match, no voltage change will be targeted NGS to make sequencing faster, more scalable, and
recorded, and no base will be called (Figure 2.2B). If there are more accessible than ever before.
two identical bases next to each other on the DNA strand, two
The semiconductor has transformed every industry it has
nucleotides are incorporated, the voltage will be double, and the
touched. Just as the microprocessor enabled desktop computing
chip will record two identical bases (Figure 2.2C).
to displace the mainframe, semiconductor technology has
Because this is direct detection—no scanning, no cameras, no the ability to democratize research. In the future, Ion Torrent
light—each nucleotide incorporation is recorded in seconds. technology may be able to provide diagnostics that are less
The process is repeated every 15 seconds with a different expensive and more reliable, helping to improve human health
nucleotide washing over the chip, and it occurs simultaneously in around the world.
millions of wells, which is why it is often described as massively
parallel sequencing.

A B C

Figure 2.2. Base calling using Ion Torrent technology. (A) As a nucleotide is added to a DNA template and incorporated into a strand of DNA, a
hydrogen ion is released, leading to a voltage change. (B) If the next nucleotide is not a match, no voltage change will be recorded. (C) Two identical
bases that are incorporated next to each other will lead to the release of two hydrogens, doubling the voltage change.

11 Contents
Contents
Chapter 3: NGS instruments—
the Ion GeneStudio S5 series
Overview
The Ion GeneStudio™ S5 systems, combined with the Ion Chef™
Ion 550™ Chip
System for automated library and template preparation, enable 100–130M reads Five chip options
a streamlined next-generation sequencing (NGS) workflow for for the GeneStudio S5 systems
enable a sequence throughput
targeted sequencing with flexibility and scalability. range of 2M to 130M reads

There are three models to choose from, giving customers options Ion 540™ Chip
60–80M reads
of different sequencing and analysis speeds at different price
points: the Ion GeneStudio™ S5 System, Ion GeneStudio™ S5 Plus
System, and Ion GeneStudio™ S5 Prime System.
Ion 530™ Chip
15–20M reads
Key features of Ion GeneStudio
S5 systems
Ion 520™ Chip
Chips 4–6M reads
The Ion GeneStudio S5 systems use sequencing chips to help
streamline workflows, while offering flexibility for a wide range of
research applications. What differentiates the chips is the amount
Ion 510™ Chip
of throughput they can deliver, ranging from 2 to 130 million 2–3M reads

reads per run. This versatility enables you to run both small‑
and large‑scale projects without the need to change platforms Figure 3.1. Throughput of the chips for Ion GeneStudio S5 systems.
(Table 3.1).

Table 3.1. Turnaround times of the Ion GeneStudio S5 systems.

Ion GeneStudio S5 Ion GeneStudio S5 Ion GeneStudio S5
System Plus System Prime System
Chip type Number of reads Read length (output)* Turnaround time (sequencing run** and analysis time)
Ion 510 Chip 2–3M 200 bp (0.3–0.5 Gb) 4.5 hr 3 hr 3 hr
400 bp (0.6–1 Gb) 10.5 hr 5 hr 5 hr
4–6M 200 bp (0.6–1 Gb) 7.5 hr 3.5 hr 3 hr
Ion 520 Chip
400 bp (1.2–2 Gb) 12 hr 5.5 hr 5.5 hr
3–4M 600 bp (0.5–1.5 Gb) 12 hr 5.5 hr 5.5 hr
Ion 530 Chip 15–20M 200 bp (3–4 Gb) 10.5 hr 5 hr 4 hr
400 bp (6–8 Gb) 21.5 hr 8 hr 6.5 hr
9–12M 600 bp (1.5–4.5 Gb) 21 hr 8 hr 7 hr
Ion 540 Chip 60–80M 200 bp (10–15 Gb) 19 hr 10 hr 6.5 hr
200 bp (20–30 Gb); NA 20 hr 10 hr †
2 runs in 1 day
Ion 550 Chip 100–130M 200 bp (20–25 Gb) NA 11.5 hr 8.5 hr
200 bp (40–50 Gb); NA NA 12 hr †
2 runs in 1 day
* Expected output with >99% aligned or measured accuracy. Output depends on read length and application.
** Sequencing run times are between 2.5 and 4 hr.
† Analysis of the first run occurs concurrently with the second sequencing run.

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Application highlights of the Ion GeneStudio S5 systems
With an extensive catalog of NGS assays covering all major Simple analysis and storage solutions
application areas, the Ion GeneStudio S5 systems are ready to Torrent Suite™ Software and Ion Reporter™ Software make NGS
meet the needs of laboratories no matter what sample comes easy for beginners and experts alike. Plan, monitor, track, and
through the door. The application areas include: analyze your runs using Torrent Suite Software (preinstalled on
the Ion PGM™ Torrent Server). Integrate, annotate, and interpret
• Cancer research—gene panels for SNPs, indels, gene
variants using Ion Reporter Software (Thermo Fisher™ Connect
expression, and gene fusion analysis
Platform or local options available based on your needs).
• Hemato-oncology research—profile for multiple relevant
driver genes in myeloid malignancies in a single test Cancer research
The Ion GeneStudio S5 systems can aid your oncology research
• Reproductive health research—aneuploidy detection
when combined with an Ion Torrent™ Oncomine™ assay design
• Inherited disease research—panels for targeted gene or that focuses on clinical research. Oncomine assays are part of an
whole-exome analysis end-to-end workflow that includes simple, scalable sequencing
with optimized bioinformatics and reporting—designed for
• Gene expression analysis—whole-transcriptome
cancer research.
RNA sequencing, targeted RNA sequencing, and small
RNA sequencing • Easily detect mutations in tumor-associated genes from
as little as 1 ng of tumor DNA with Ion AmpliSeq™ cancer
• Microbiology and infectious disease research—
research gene panels, or create custom assays with the online
SARS‑CoV-2, microbial whole genomes, microbial Ion AmpliSeq™ Designer tool
genotyping, and metagenomics
• Identify single-nucleotide variants (SNVs), indels, copy
• Microbiome research—profiling of microbial diversity in the number variants (CNVs), and gene fusions starting from
human gut microbiome precious clinical research samples using our growing menu of
Oncomine assays for precision oncology research, such as the
Small sample input Ion Torrent™ Oncomine™ Comprehensive Assay Plus
Low input DNA from challenging sample types such as
• Perform analysis on small, archived FFPE solid tumor and fine
formalin-fixed, paraffin-embedded (FFPE) tissue, retrospective needle aspirate samples
samples from fine needle aspirates, and cell-free DNA
(cfDNA) extracted from blood can be difficult to sequence
on next-generation sequencers from other suppliers. With
Ion AmpliSeq™ technology and the Ion GeneStudio S5 series
instruments, you can use as little as 1 ng of input DNA or RNA.

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Complex and inherited disease research Microbiology and infectious disease research
The Ion GeneStudio S5 systems deliver an end-to-end NGS Uncover microbial diversity, study pathogen outbreaks, and
solution for researchers studying complex and inherited identify mutations that may be associated with antibiotic
diseases. Using Ion AmpliSeq targeted assay technology, resistance. Take advantage of high throughput and accuracy
thousands of amplicons, including the complete exome, can be with long reads to produce rapid and accurate sequencing of
rapidly amplified by PCR, followed by sequencing using the Ion microbes with streamlined sample preparation and a simple,
GeneStudio S5 System. Ready-to-use and customizable Ion scalable, and optimized workflow for data analysis.
AmpliSeq assays are available to target genetic loci of interest
• Conduct rapid and affordable bacterial and viral typing
for inherited disease research, including hereditary cancers,
research during disease surveillance, using archived samples
neurodegenerative disorders, and cardiovascular diseases. from investigations and disease etiology studies, such as
Primer pools can also be created to target any customer-defined studies of SARS-CoV-2, MERS, H1N1, and Ebola.
region of interest within a reference sequence.
• Study microbial communities and conduct metagenomic
• Predesigned assays support cost-effective detection of single- research leveraging 16S rRNA sequencing or other
nucleotide variants (SNVs), indels, and copy number variants targeted panels with Ion AmpliSeq technology. Based
(CNVs) for inherited disease research. on ultrahigh-multiplex PCR, Ion AmpliSeq technology
requires as little as 1 ng of input DNA and uses a simple,
• Customize existing inherited disease research panel designs streamlined workflow.
at the gene, region, or amplicon level, and deliver just the
content needed by leveraging the simple-to-use Ion AmpliSeq
Designer tool.

Reproductive health research

Understanding risk factors for inherited genetic disorders is
an important step in evaluating embryos. Expanded carrier
screening (ECS) research and preimplantation genetic testing
(PGT) offer scientific insights that may increase probability of
success for future pregnancies and healthier future generations.
Ion GeneStudio S5 systems facilitate scalable, precise
sequencing solutions for ECS and PGT research.

• Comprehensive ECS for research of a broad range of

inherited disorders—The Ion Torrent™ CarrierSeq™ ECS Kit
contains a 420-gene panel that targets full coding regions,
enabling the analysis of >36,000 non-benign ClinVar variants
for SNVs, indels, and CNVs by NGS.

• Combined PGT from a single biopsy—The Ion Torrent™

ReproSeq™ PGS kit for PGT-A (aneuploidy) and Ion Torrent™
PGD-SEQ™ kit for PGT-M (monogenic disease) can be used
to simultaneously study chromosomal abnormalities and
variants linked to single-gene disorders, in one convenient
NGS workflow.

14 Contents
Conclusion
The ability of the Ion GeneStudio S5 systems to work with multiple read lengths helps
you achieve fast and consistent results irrespective of the size of your project. You can
save your precious samples by using quantities as low as picograms and nanograms.
The Ion GeneStudio S5 systems are great for customers who are seeking flexibility
and a wide array of application options. In the next chapter, you will learn about the Ion
Torrent™ Genexus™ System, which uses the same sophisticated sequencing technology
as the Ion GeneStudio S5 systems but delivers an advanced level of automation and
ease of use.

15 Contents
Chapter 4: NGS instruments—
the Genexus System
Overview
The Ion Torrent™ Genexus™ System automates sample and
library/template preparation, sequencing, analysis, and reporting.
The Ion Torrent™ Genexus™ Purification System and Genexus™
Integrated Sequencer with Genexus™ Software work together
seamlessly, tracking sample information and results automatically
throughout the process.

This integrated system can deliver an end-to-end workflow • The Genexus Integrated Sequencer automates library
from the biological specimen all the way to the report in a single preparation, templating, and sequencing.
day with just two user touchpoints and 20 minutes of total • The Genexus Software fully integrates the purification system
hands-on time. with the sequencer, reducing the possibility of human error
and making reporting simple. Data files can be exported for
• The Genexus Purification System extracts and quantifies third-party analytics, or analytic tools from Thermo Fisher
nucleic acids in approximately 2–4 hours. Scientific can be used to generate customizable report formats
based on guidelines, clinical trials, curated markers, and
novel variants.

The Genexus Purification System

The Genexus Purification System automates nucleic acid Innovative features of the Genexus Purification
extraction, purification, and quantitation on a single platform System include:
to enable a consistent and efficient workflow solution for • Easy operation and automated setup for error detection—
next-generation sequencing (NGS) sample preparation. Work simply with prefilled reagents that require only one
touchpoint and confirm correct reagent placement with
Extract, purify, and quantify nucleic acid samples— onscreen guidance for loading consumables onto the
instrument. The automated barcode scanning helps identify
all on a single automated platform
consumable expiration dates and detect errors.
The system helps minimize user processing errors and increases
the reproducibility of your results by requiring just one touchpoint • Verified protocols—Produce reliable data for downstream

with 10 minutes of hands-on time. In as little a 2 hours, the NGS on the Genexus Integrated Sequencer.

system will provide you with a plate of purified and quantified • Rapid turnaround—Go from specimen to purified and
nucleic acid sample that is ready to be loaded onto the Genexus quantified nucleic acid that is ready for NGS analysis in as little
Integrated Sequencer for downstream applications. The Genexus as 2 hours.
Purification System supports multiple specimen types to enable • Manufactured at an FDA-registered and
NGS applications (Table 4.1). ISO 13485–certified facility—Have total confidence in the
quality of your instrument.

Table 4.1. Ion Torrent™ Genexus™ kits for sample preparation.

Kit Specimen type Output nucleic acid Reactions per kit
Genexus FFPE DNA and RNA Lysate from FFPE tissue (surgical DNA and RNA, DNA only, 48
Purification Kit (sequential resection, core needle biopsy, and or RNA only
extraction of DNA and RNA) fine needle aspiration)
Genexus Cell-Free Total Nucleic Plasma cfTNA 24
Acid Purification Kit
Genexus Multisample DNA Whole blood, PBLs, lysate from DNA only 48
Purification Kit fresh-frozen tissue, and bone
marrow
Genexus Total RNA Purification Whole blood, PBLs, lysate from RNA only 48
Kit fresh-frozen tissue, and bone
marrow

16 Contents
The Genexus Integrated Sequencer Application highlights of the
The Genexus System enables a workflow from a biological Genexus System
specimen all the way to the final report, with results delivered Learn how scientists, clinical researchers, and other laboratory
in as little as a single day. The Genexus Integrated Sequencer personnel use automated workflows and the Genexus System to
can also be used as a stand-alone instrument to automate NGS complete NGS.
library preparation, sequencing, and analysis on a single platform.

Using the multilane Ion Torrent™ GX5™ Chip, which is designed Oncology clinical research
to support variability in sample intake, samples can be Ion Torrent™ Oncomine™ assays for the Genexus System

cost-effectively processed as they come in. feature a complete NGS testing workflow. Now every laboratory
can go from specimen to report in a single day, providing a
Innovative features of the Genexus Integrated Sequencer: comprehensive genomic profile. This allows you to deliver NGS
results at the same time as other single-gene methods, such as
• Hands-off automation—Library prep, sequencing, and
analysis all happen on one instrument with a simple set-up- immunohistochemistry (IHC).
and-go workflow.
The limited hands-on, set-up-and-go workflow makes NGS
• Streamlined on-instrument analysis—Integrated reporting accessible even if your laboratory is new to the technology.
capabilities are supported, no server required. With the lowest sample input requirement of any NGS solution,
• Easy operation—The instrument uses prefilled reagents you can successfully sequence more of the samples that come
and preset protocols, a single touchpoint, and 10 minutes of through your laboratory.
hands-on time.*

• Rapid turnaround—Go from nucleic acid to report in as little

as a single day.**

• Flexibility to accommodate small sample batches—

On-instrument reagent and chip stability supports sample
intake variability.

• Onboard vision system—Understand reagent placement and

detect errors through automated barcode scanning.

• Manufactured at an FDA-registered and ISO

13485–certified facility—Have total confidence in your
instrument’s quality.

• Innovative multilane chip—Simultaneously process up to four

compatible assays in a single run.

* 10 minutes of hands-on time when running in integrated mode with the Genexus Purification System. A few
more minutes to accommodate pipetting step time are needed when using as a stand-alone instrument.
** Processing times for individual samples vary.

17 Contents
 Day 1 Day 2
8 a.m. 4 p.m. 12 a.m. 8 a.m. 4 p.m.

Start with Get variant

viral RNA report

Microbial and infectious disease research

Automated cDNA synthesis, library
Ion AmpliSeq™ targeted NGS panels run on the Genexus prep, template prep, sequencing,
Integrated Sequencer provide an ultrasensitive, rapid, and and analysis

accessible solution for infectious disease researchers. The panels

provide a fast and accurate platform for sequencing of microbial
Figure 4.1. A typical workflow for analyzing 16 samples using the Ion
genomes, suitable for bacterial and viral typing, epidemiological
AmpliSeq SARS-CoV-2 panel on the Genexus Integrated Sequencer,
studies, and disease surveillance. featuring rapid NGS automation.

Benefits of using targeted NGS and the Genexus Integrated

Sequencer for studying infectious diseases include: Through the use of a set of highly specific, universal coronavirus
primers in combination with a high-fidelity master mix, all
• Rapid turnaround time—our fastest NGS workflow for
infectious disease research for time-sensitive applications genomic segments are amplified, sequenced, and analyzed in
an automated workflow on the Genexus Integrated Sequencer.
• Automation—the set-up-and-go workflow enables minimal
This NGS solution enables you to rapidly go from nucleic acid to
user touchpoints, which is designed to reduce user variability
variant report in under a single day with minimal hands-on time
and increase the reproducibility of results
(Figure 4.1).
• Accuracy of variants—lower substitution errors for single-
nucleotide variants (SNVs) The Ion AmpliSeq™ SARS-CoV-2 Insight Research Assay
consists of two pools with amplicons 125–275 bp in length for
• Low input requirements—need as little as 1 ng of input
nucleic acid to target regions of interest complete coverage of over 99% of the SARS-CoV-2 genome,
including all variants [1].
• Custom panel design options—customize your own
infectious disease research panel using the Ion AmpliSeq
Designer tool at ampliseq.com

Coronavirus (SARS-CoV-2) research

A major challenge for microbiologists and virologists is the
prediction of patterns of evolution and emergence of disease
agents. RNA viruses like coronaviruses share the feature of high
genetic variability, which causes them to phylogenetically cluster
as clouds of mutants. Coronavirus variants also emerge through
antigenic shifts within animal reservoirs, such as bats, snakes,
and pangolins.

18 Contents
Benefits of the Ion AmpliSeq SARS-CoV-2 Insight Inherited disease clinical research
Research Assay for the Genexus Integrated Since 2011, Ion Torrent products for NGS have supported
Sequencer include: clinical research of human diseases with leading
• Rapid turnaround time—our fastest NGS Ion AmpliSeq technology.
workflow for infectious disease research for critically
time-sensitive applications Now, Ion AmpliSeq™ On-Demand Panels are available for the
Genexus System. Together, they provide a powerful and targeted
• Automation—the set-up-and-go workflow enables minimal
NGS workflow that enables any laboratory to go from specimen
user touchpoints, reducing user variability and increasing
to variant report in a single day, providing accessibility, efficiency,
reproducibility of results
and convenience at a speed never possible before (Figure 4.2).
• Accuracy of variants—fewer substitution errors for SNVs

• Higher success rates—directly analyze samples that have low

viral loads

• Higher-resolution and longer-read NGS—accurate and rapid

virus typing for surveillance and epidemiology investigations

Ion Torrent™ Genexus™ Software

Genexus Genexus
Purification System Integrated Sequencer

• Whole blood Automated Automated

• Plasma nucleic acid library/template
extraction and preparation,
purification sequencing, and
variant analysis

Figure 4.2. Workflow for specimen to variant report using the Genexus System.
* Specimen-to-variant report workflow will be available after the Ion Torrent Genexus Purification System and integrated reporting
capabilities are added in 2020.

19 Contents
The Genexus System integrates and
automates nucleic acid extraction and
purification, library/template preparation,
sequencing, and analysis in a single
software ecosystem. This highly flexible
system lets you process samples—even
just one—cost-effectively as they come
in. With just 20 minutes of hands-on time
and two touchpoints, users can get up
and running quickly with significantly less
training. The set-up-and-go workflow
makes NGS accessible, even if your
laboratory is new to the technology.

20 Contents
Conclusion
Next-generation sequencing is a powerful tool that has a wide variety of research
applications. Targeted sequencing can be used to identify variations associated with
individual organisms, genes, or gene-fragments; these variations can inform our
understanding of disease mechanisms and immunity to infectious diseases, as well
as the role of genes and gene variants in inherited diseases. Ion Torrent technology
is accessible to laboratories of nearly any size and budget, and can be purchased
alongside a full set of reagents, assays, and consumables from Thermo Fisher Scientific.

Chapter 4 reference
1. Thermo Fisher Scientific. Ion AmpliSeq SARS-CoV-2 Insight Research Assay. https://www.thermofisher.com/us/en/
home/life-science/sequencing/dna-sequencing/microbial-sequencing/microbial-identification-ion-torrent-
next-generation-sequencing/viral-typing/coronavirus-research.html.

Learn more at thermofisher.com/ngsebook

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