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bcl2fastq on Biowulf

The primary output of Illumina sequencing instruments are per-cycle base call files in BCL format. Bcl2fastq converts BCL files to fastq files used by most downstream software. It also separates reads into individual fastq files based on their barcode (demultiplexing), does adapter masking/trimming and moves unique molecular identifier (UMI) bases from the read to the fastq header. Bcl2fastq operates on Illumina run folders.

bcl2fastq creates a number of files summarizing statistics of the conversion in the InterOp folder of the run folder. They can be examined with the Illumina Sequence Analysis Viewer.

Important Notes

bcl2fastq is a multithreaded application. By default it will run as many threads as there are CPUs for the conversion/demultiplexing plus additional threads for reading/writing data. You must therefore limit the threads to the number of threads allocated to your job or allocate nodes exclusively.

The relevant options for limiting the number of threads are

-r [ --loading-threads ] arg (=4)     number of threads used for loading BCL data
-d [ --demultiplexing-threads ] arg   number of threads used for demultiplexing
-p [ --processing-threads ] arg       number of threads used for processing demultiplexed data
-w [ --writing-threads ] arg (=4)     number of threads used for writing FASTQ data

For a job allocated 16 CPUs these options should be set to

bcl2fastq -r 2 -w 2 -d 4 -p 14 ...

This leads to a nominal overload but according to testing by Illumina this should yield optimal throughput.

Interactive job
Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --cpus-per-task=16
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144 ~]$ module load bcl2fastq
[user@cn3144 ~]$ bcl2fastq --help
BCL to FASTQ file converter
bcl2fastq v2.20.0.422
Copyright (c) 2007-2017 Illumina, Inc.

      bcl2fastq [options]

[user@cn3144 ~]$ bcl2fastq -r 2 -w 2 -d 4 -p 14 \
    --runfolder-dir /path/to/your/run/folder/160601_mach1_0023 ...

[user@cn3144 ~]$

[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

Batch job
Most jobs should be run as batch jobs.

Create a batch input file (e.g. bcl2fastq.sh), which uses the input file 'bcl2fastq.in'. For example:

#! /bin/bash

module load bcl2fastq/2.20.0 || exit 1
bcl2fastq --runfolder-dir /path/to/your/run/folder/160601_mach1_0023 \
          --output-dir ./160601_mach1_0023 \
          -r 2 -w 2 -d 4 -p 14 \
          --barcode-mismatches 0

Submit this job using the Slurm sbatch command.

sbatch --cpus-per-task=16 bcl2fastq.sh
Swarm of Jobs
A swarm of jobs is an easy way to submit a set of independent commands requiring identical resources.

Create a swarmfile (e.g. bcl2fastq.swarm). For example:

bcl2fastq --runfolder-dir /path/to/your/run/folder/160601_mach1_0023 \
          --output-dir ./160601_mach1_0023 \
          -r 2 -w 2 -d 4 -p 14
bcl2fastq --runfolder-dir /path/to/your/run/folder/160602_mach1_0024 \
          --output-dir ./160602_mach1_0024 \
          -r 2 -w 2 -d 4 -p 14
bcl2fastq --runfolder-dir /path/to/your/run/folder/160603_mach1_0025 \
          --output-dir ./160603_mach1_0025 \
          -r 2 -w 2 -d 4 -p 14

Submit this job using the swarm command.

swarm -f bcl2fastq.swarm -t 16 --module bcl2fastq
-t # Number of threads/CPUs required for each process (1 line in the swarm command file).
--module bcl2fastq Loads the bcl2fastq module for each subjob in the swarm