Example and tutorial for SneakerNet

This directory contains an example for how you would use SneakerNet. There is an 'inbox' and a Rover Spreadsheet. First, this document will describe Rover; second, this document will describe how to use SneakerNet.

The Rover spreadsheet

Rover is a spreadsheet that can explore your reads and return basic information for you. This is a double meaning between the NASA planatary vehicles and a dog that might fetch for you! This example will not show you how to perform a sequencing run because the wet lab is outside of the scope of this project. However, this example will show you how to fill in the appropriate sample spreadsheet for the MiSeq, which SneakerNet will also read.

How to use the Rover spreadsheet

There are two rover spreadsheet examples in this directory, and they differ only on the number of samples.

• Rover_NexteraXTv2_16samples.xlsx
• Rover_NexteraXTv2_48samples.xlsx

Thank you to the National Enteric Reference Laboratory (NERL) for the basis of this documentation and spreadsheets.

1. Ensure that the full header (i.e., "Run Name", "Sample Plate Name", "Sample Sheet Name", "Library Prep Date", and "Technician" fields) and sample-related fields for each sample (i.e., "Sample Name", "Organism", "Index 1", "Index 2", and "Genome Size Estimate") are filled in on the "Initial Dilution" tab.

2. Optional: If raw read files are to be automatically transferred from the storage space to another location (e.g., Calculation Engine of BioNumerics), then enter "Yes" in the appropriate column for each sample to transfer on the "Raw Read Routing" tab.

3. Navigate to the "SampleSheet" tab of the Library Prep Workbook, and "Save As..." a "CSV (Comma delimited) (*.csv)" file on a USB portable hard drive.

• Note: Because the workbook contains several tabs, a message might pop up along the lines of "The selected file type does not support workbooks that contain multiple sheets..." Click OK to save only the SampleSheet tab.
  
• Excel will continue to ask you the OK/Cancel question about there being multiple sheets until you click Cancel. The file is saved where you specify when you click OK the first time, so hit Cancel when prompted the second time.
  
• Then, because the SampleSheet tab contains formulas, a second warning will pop up "Some features in your workbook might be lost... Do you want to keep using that format?" Click No, and the file will be auto-saved to your selected drive. (See warning above about second OK/Cancel prompt)
  

4. On the MiSeq, open the Illumina Experiment Manager, and choose "Edit Sample Sheet"
   
5. Navigate to your sample sheet csv file, and make sure all relevant fields are filled in, including the "Use Adapter Trimming" checkbox on the right side of the screen, and all indices used are present and compatible.

Using SneakerNet

If you have installed SneakerNet correctly including editing the config files, then all you need to do is run it on the simulated example in the inbox folder.

Use --help to get SneakerNet help. You can also use --help on any SneakerNet plugin to get help.

$ SneakerNet.pl --help
$ addReadMetrics.pl --help

To run SneakerNet on the example, use this syntax:

$ SneakerNet.pl --inbox example/inbox --now

Using the --now flag assumes that no one can modify the run directory before you use it. Without this flag, SneakerNet monitors the inbox for two minutes to be sure that no one is currently adding a run, so that the run does not get corrupted. Therefore you should not use the --now flag in everyday use when others have access to the inbox.

Requirements for a run

Currently, SneakerNet can only be used on Illumina-based run directories. The directory name must be in the form of machineName-year-runNumber-comment where machine name is a custom name, e.g., M0347 for a MiSeq. The year is a 2 or 4-digit year. The run number is an integer. The comment is optional but can be used to help indicate the date of a run or whatever else. Collectively, machineName-year-runNumber-comment is the runId that might be referred elsewhere in the documentation.

The following files must be present in a run directory before SneakerNet will accept it.

• QC information
  • QC - this is a folder that you create manually
  • QC/runInfo.xml
  • QC/runParameters.xml
  • QC/CompletedJobInfo.xml
  • QC/GenerateFASTQRunStatistics.xml
  • QC/InterOp/ - this is a folder from an Illumina run with QC information. Keep all files in it intact.
• Data and metadata
  • *.fastq.gz
  • SampleSheet.csv, usually derived from Rover
  • config.xml

Optional files for a run

At least one file at this time is optional

• snok.txt
  • Its presence helps SneakerNet understand that a run is ready to be read by SneakerNet. It can be present in the root directory of a full run instead of a folder with extracted contents.
  • It can also have key value pairs. This file is still being developed and can accept the following key/value pairs, without ini-style headers.
    • emails = [email protected], [email protected], ...
    • workflow = default
      • can also be metagenomics or iontorrent