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Bcl2fastq2 For NextSeq

and HiSeq X Systems

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By the end of this training you will:

– Know how to obtain bcl2fastq2

– Create a SampleSheet to be used by bcl2fastq

– Understand the bcl2fastq workflow for the NextSeq 500 and/or HiSeq X

– Be able to perform basic de-multiplexing troubleshooting

Session Objectives

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Download Bcl2fastq2

http://support.illumina.com/downloads/bcl2fastq_conversion_software.html

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Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

Workflow

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Select Sequencer and Click Next

HiSeq X

Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

NextSeq

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Select FastQ Only and Click Next

Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

HiSeq X NextSeq

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Select Workflow Parameters

Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

SampleSheet must be entered in reagent

barcode field in order for bcl2fastq2 to

recognize the sample sheet.

8

Add Samples and Select Indices

Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

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Exercise: Illumina Experiment Manager for

NextSeq

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Reviewing the Sample Sheet

Open in Excel immediately after

sample sheet creation within IEM

Review Sample Sheet on Linux Analysis Server

immediately before running bcl2fastq2

1) Navigate to run folder

2) Use command cat SampleSheet.csv to view

in terminal

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Analysis Server Requirements

Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

NextSeq HiSeq X The BCL to FASTQ converter

needs the following input files :

• BCL files (*.bcl.bgzf) or (bcl.gz)

• *.bci files

• *.filter files

• *.locs or s.locs file

• RunInfo.xml file.

• (Optionally) SampleSheet.csv.

Reminder: Sample sheet

must be named SampleSheet.csv

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Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

Open PUTTY.EXE

Enter your Server Details

Login to Server

Open Terminal Session on Analysis Server

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Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

Invoke bcl2fastq in the following way:

1) Navigate to the root run folder by using the cd command

2) Use the command nohup /usr/local/bin/bcl2fastq

3) When process completes, command line reappears

A Few Helpful Linux Commands and options

Step 1

Steps 2 & 3

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Command Usage

cd To change directory

ls -alh Lists all files in directory in human readable format with sizes

cat Displays the contents of a file (use with small files)

zcat Displays the contents of a compressed file

Head [option] Shows the first specified number of lines in a file

cd .. Moves up one level in the directory structure

/usr/local/bin/bcl2fastq [options] Invokes bcl2fastq (See user guide for all available options)

A Few Helpful Linux Commands and Options

Options Usage

& Allows script to run in the background (placed at end of line)

nohup Closing the terminal session will not terminate process

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Use IEM to create

Sample Sheet

Place Data on Analysis Server

Putty into Analysis Server

Run bcl2fastq2

View fastqfiles

View Completed FastQ files

View FastQ directly on Linux system by

navigating to

Runfolder>Data>Intensities>BaseCalls*

*or location specified by option -- output-dir

View FastQ files in terminal by navigating to the

BaseCalls folder and using the command ls –alh

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Troubleshooting Demultiplexing

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Bcl2fastq2 Demultiplexing Scenarios

Is SampleSheet.csv present in run folder?

Or sample sheet does not contain [Data] Section

All reads placed in default Undetermined FASTQ files by lane

Are indexes provided in Sample sheet?

All reads placed in sample FASTQ files

Do reads match defined indexes?

Reads with no valid index placed in Undetermined_S0 FASTQ files

Reads with valid indexes placed in FASTQ files as defined in the sample sheet.

If project is defined in sample sheet, converter will

output fastq files to project folders

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Bcl2fastq2 Resources

C

Converter User Guide and Release

Notes available on website

http://support.illumina.com/downloads/

bcl2fastq_conversion_software.html

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Questions?