Biowulf High Performance Computing at the NIH
freebayes on Biowulf

FreeBayes is a Bayesian genetic variant detector designed to find small polymorphisms, specifically SNPs (single-nucleotide polymorphisms), indels (insertions and deletions), MNPs (multi-nucleotide polymorphisms), and complex events (composite insertion and substitution events) smaller than the length of a short-read sequencing alignment.

FreeBayes is haplotype-based, in the sense that it calls variants based on the literal sequences of reads aligned to a particular target, not their precise alignment. This model is a straightforward generalization of previous ones (e.g. PolyBayes, samtools, GATK) which detect or report variants based on alignments. This method avoids one of the core problems with alignment-based variant detection--- that identical sequences may have multiple possible alignments


Important Notes

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 --mem=10g
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 freebayes samtools
[user@cn3144 ~]$ freebayes \
    --fasta-reference /fdb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa \
    --min-alternate-count 2 \
    --min-alternate-qsum 40 \
    --pvar 0.0001 \
    --use-mapping-quality \
    --site-selection-max-iterations 3 \
    --genotyping-max-iterations 25 \
    /fdb/app_testdata/bam/hg19/gcat_set_053.bam \
  | bgzip -c \
  > test.vcf.gz

# This takes approximately 72 minutes.

[user@cn3144 ~]$ bgzip -r test.vcf.gz
[user@cn3144 ~]$ ls -lh
-rw-r--r-- 1 user group 30M Feb 12 14:14 test.vcf.gz
-rw-r--r-- 1 user group 48K Feb 12 14:17 test.vcf.gz.gzi
[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., which uses the input file ''. For example:

#! /bin/bash

module load freebayes/1.1.0 samtools || exit 1
freebayes --fasta-reference /fdb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa \
    --min-alternate-count 2 \
    --min-alternate-qsum 40 \
    --pvar 0.0001 \
    --use-mapping-quality \
    --site-selection-max-iterations 3 \
    --genotyping-max-iterations 25 \
    secret_sample_1.bam \
  | bgzip -c \
  > secret_sample_1.vcf.gz

Submit this job using the Slurm sbatch command.

sbatch --cpus-per-task=2 --mem=10g
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. freebayes.swarm). For example:

cd /data/$USER/dir1; freebayes --fasta-reference ../fa/h.sapiens.fasta sample1.bam | bgzip -c > sample1.vcf.gz
cd /data/$USER/dir1; freebayes --fasta-reference ../fa/h.sapiens.fasta sample2.bam | bgzip -c > sample2.vcf.gz
cd /data/$USER/dir1; freebayes --fasta-reference ../fa/h.sapiens.fasta sample3.bam | bgzip -c > sample3.vcf.gz

Submit this job using the swarm command.

swarm -f freebayes.swarm -g 10 --module freebayes/1.1.0,samtools
-g # Number of Gigabytes of memory required for each process (1 line in the swarm command file)
-t # Number of threads/CPUs required for each process (1 line in the swarm command file).
--module freebayes Loads the freebayes module for each subjob in the swarm