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

References:

• Erik Garrison, Gabor Marth. Haplotype-based variant detection from short-read sequencing. 2012, arXiv:1207.3907

• freebayes GitHub repo

• Module Name: freebayes (see the modules page for more information)
• freebayes can be parallelized with freebayes-parallel
• Example data in $FREEBAYES_TEST_DATA

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 ~]$

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

#! /bin/bash

module load freebayes 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 freebayes.sh

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