What it does
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.
See https://github.com/ekg/freebayes for details on FreeBayes.
Description
Provided some BAM dataset(s) and a reference sequence, FreeBayes will produce a VCF dataset describing SNPs, indels, and complex variants in samples in the input alignments.
By default, FreeBayes will consider variants supported by at least 2 observations in a single sample (-C) and also by at least 20% of the reads from a single sample (-F). These settings are suitable to low to high depth sequencing in haploid and diploid samples, but users working with polyploid or pooled samples may wish to adjust them depending on the characteristics of their sequencing data.
FreeBayes is capable of calling variant haplotypes shorter than a read length where multiple polymorphisms segregate on the same read. The maximum distance between polymorphisms phased in this way is determined by the --max-complex-gap, which defaults to 3bp. In practice, this can comfortably be set to half the read length.
Ploidy may be set to any level (-p), but by default all samples are assumed to be diploid. FreeBayes can model per-sample and per-region variation in copy-number (-A) using a copy-number variation map.
FreeBayes can act as a frequency-based pooled caller and describe variants and haplotypes in terms of observation frequency rather than called genotypes. To do so, use --pooled-continuous and set input filters to a suitable level. Allele observation counts will be described by AO and RO fields in the VCF output.
Galaxy-specific options
Galaxy allows five levels of control over FreeBayes options, provided by the Choose parameter selection level menu option. These are:
- Simple diploid calling: The simplest possible FreeBayes application. Equivalent to using FreeBayes with only a BAM input and no other parameter options.
- Simple diploid calling with filtering and coverage: Same as #1 plus two additional options: -0 (standard filters: --min-mapping-quality 30 --min-base-quality 20 --min-supporting-allele-qsum 0 --genotype-variant-threshold 0) and --min-coverage.
- Frequency-based pooled calling: This is equivalent to using FreeBayes with the following options: --haplotype-length 0 --min-alternate-count 1 --min-alternate-fraction 0 --pooled-continuous --report-monomorphic. This is the best choice for calling variants in mixtures such as viral, bacterial, or organellar genomes.
- Frequency-based pooled calling with filtering and coverage: Same as #3 but adds -0 and --min-coverage like in #2.
- Complete list of all options: Gives you full control by exposing all FreeBayes options as Galaxy parameters.
Command-line parameters
Input:
--bam FILE The file or set of BAM files to be analyzed.
--bam-list FILE A file containing a list of BAM files to be analyzed.
--stdin Read BAM input on stdin.
--fasta-reference FILE Use FILE as the reference sequence for analysis.
An index file (FILE.fai) will be created if none exists.
If neither --targets nor --region are specified, FreeBayes
will analyze every position in this reference.
--targets FILE Limit analysis to targets listed in the BED-format FILE.
--region <chrom>:<start>-<end> Limit analysis to the specified region, 0-base coordinates,
end_position not included (same as BED format).
Either '-' or '..' maybe used as a separator.
--samples FILE Limit analysis to samples listed (one per line) in the FILE.
By default FreeBayes will analyze all samples in its input
BAM files.
--populations FILE Each line of FILE should list a sample and a population which
it is part of. The population-based bayesian inference model
will then be partitioned on the basis of the populations.
--cnv-map FILE Read a copy number map from the BED file FILE, which has
either a sample-level ploidy:
sample_name copy_number
or a region-specific format:
seq_name start end sample_name copy_number
... for each region in each sample which does not have the
default copy number as set by --ploidy. These fields can be delimited
by space or tab.
Output:
--vcf FILE Output VCF-format results to FILE. (default: stdout)
--gvcf Write gVCF output, which indicates coverage in uncalled regions.
--gvcf-chunk NUM When writing gVCF output emit a record for every NUM bases.
--gvcf-dont-use-chunk When writing the gVCF output emit a record for all bases if
set to "true" , will also route an int to --gvcf-chunk
similar to --output-mode EMIT_ALL_SITES from GATK
--variant-input VCF Use variants reported in VCF file as input to the algorithm.
Variants in this file will included in the output even if
there is not enough support in the data to pass input filters.
--only-use-input-alleles Only provide variant calls and genotype likelihoods for sites
and alleles which are provided in the VCF input, and provide
output in the VCF for all input alleles, not just those which
have support in the data.
--haplotype-basis-alleles VCF When specified, only variant alleles provided in this input
VCF will be used for the construction of complex or haplotype
alleles.
--report-all-haplotype-alleles At sites where genotypes are made over haplotype alleles,
provide information about all alleles in output, not only
those which are called.
--report-monomorphic Report even loci which appear to be monomorphic, and report all
considered alleles, even those which are not in called genotypes.
Loci which do not have any potential alternates have '.' for ALT.
--pvar N Report sites if the probability that there is a polymorphism
at the site is greater than N. default: 0.0. Note that post-
filtering is generally recommended over the use of this parameter.
--strict-vcf Generate strict VCF format (FORMAT/GQ will be an int)
Population model:
--theta N The expected mutation rate or pairwise nucleotide diversity
among the population under analysis. This serves as the
single parameter to the Ewens Sampling Formula prior model
default: 0.001
--ploidy N Sets the default ploidy for the analysis to N. default: 2
--pooled-discrete Assume that samples result from pooled sequencing.
Model pooled samples using discrete genotypes across pools.
When using this flag, set --ploidy to the number of
alleles in each sample or use the --cnv-map to define
per-sample ploidy.
--pooled-continuous Output all alleles which pass input filters, regardles of
genotyping outcome or model.
Reference allele:
--use-reference-allele This flag includes the reference allele in the analysis as
if it is another sample from the same population.
--reference-quality MQ,BQ Assign mapping quality of MQ to the reference allele at each
site and base quality of BQ. default: 100,60
Allele scope:
--use-best-n-alleles N Evaluate only the best N SNP alleles, ranked by sum of
supporting quality scores. (Set to 0 to use all; default: all)
--max-complex-gap
--haplotype-length N Allow haplotype calls with contiguous embedded matches of up
to this length. Set N=-1 to disable clumping. (default: 3)
--min-repeat-size When assembling observations across repeats, require the total repeat
length at least this many bp. (default: 5)
--min-repeat-entropy N To detect interrupted repeats, build across sequence until it has
entropy > N bits per bp. Set to 0 to turn off. (default: 1)
--no-partial-observations Exclude observations which do not fully span the dynamically-determined
detection window. (default, use all observations, dividing partial
support across matching haplotypes when generating haplotypes.)
Indel realignment:
--dont-left-align-indels Turn off left-alignment of indels, which is enabled by default.
Input filters:
--use-duplicate-reads Include duplicate-marked alignments in the analysis.
default: exclude duplicates marked as such in alignments
--min-mapping-quality Q Exclude alignments from analysis if they have a mapping
quality less than Q. default: 1
--min-base-quality Q Exclude alleles from analysis if their supporting base
quality is less than Q. default: 0
--min-supporting-allele-qsum Q Consider any allele in which the sum of qualities of supporting
observations is at least Q. default: 0
--min-supporting-mapping-qsum Q Consider any allele in which and the sum of mapping qualities of
supporting reads is at least Q. default: 0
--mismatch-base-quality-threshold Q Count mismatches toward --read-mismatch-limit if the base
quality of the mismatch is >= Q. default: 10
--read-mismatch-limit N Exclude reads with more than N mismatches where each mismatch
has base quality >= mismatch-base-quality-threshold.
default: ~unbounded
--read-max-mismatch-fraction N Exclude reads with more than N [0,1] fraction of mismatches where
each mismatch has base quality >= mismatch-base-quality-threshold
default: 1.0
--read-snp-limit N Exclude reads with more than N base mismatches, ignoring gaps
with quality >= mismatch-base-quality-threshold.
default: ~unbounded
--read-indel-limit N Exclude reads with more than N separate gaps.
default: ~unbounded
--standard-filters Use stringent input base and mapping quality filters
Equivalent to -m 30 -q 20 -R 0 -S 0
--min-alternate-fraction N Require at least this fraction of observations supporting
an alternate allele within a single individual in the
in order to evaluate the position. default: 0.05
--min-alternate-count N Require at least this count of observations supporting
an alternate allele within a single individual in order
to evaluate the position. default: 2
--min-alternate-qsum N Require at least this sum of quality of observations supporting
an alternate allele within a single individual in order
to evaluate the position. default: 0
--min-alternate-total N Require at least this count of observations supporting
an alternate allele within the total population in order
to use the allele in analysis. default: 1
--min-coverage N Require at least this coverage to process a site. default: 0
--limit-coverage N Downsample per-sample coverage to this level if greater than this coverage.
default: no limit
--skip-coverage N Skip processing of alignments overlapping positions with coverage >N.
This filters sites above this coverage, but will also reduce data nearby.
default: no limit
Population priors:
--no-population-priors Equivalent to --pooled-discrete --hwe-priors-off and removal of
Ewens Sampling Formula component of priors.
Mappability priors:
--hwe-priors-off Disable estimation of the probability of the combination
arising under HWE given the allele frequency as estimated
by observation frequency.
--binomial-obs-priors-off Disable incorporation of prior expectations about observations.
Uses read placement probability, strand balance probability,
and read position (5'-3') probability.
--allele-balance-priors-off Disable use of aggregate probability of observation balance between alleles
as a component of the priors.
Genotype likelihoods:
--observation-bias FILE Read length-dependent allele observation biases from FILE.
The format is [length] [alignment efficiency relative to reference]
where the efficiency is 1 if there is no relative observation bias.
--base-quality-cap Q Limit estimated observation quality by capping base quality at Q.
--prob-contamination F An estimate of contamination to use for all samples. default: 10e-9
--legacy-gls Use legacy (polybayes equivalent) genotype likelihood calculations
--contamination-estimates FILE A file containing per-sample estimates of contamination, such as
those generated by VerifyBamID. The format should be:
sample p(read=R|genotype=AR) p(read=A|genotype=AA)
Sample '*' can be used to set default contamination estimates.
Algorithmic features:
--report-genotype-likelihood-max Report genotypes using the maximum-likelihood estimate provided
from genotype likelihoods.
--genotyping-max-iterations N Iterate no more than N times during genotyping step. default: 1000.
--genotyping-max-banddepth N Integrate no deeper than the Nth best genotype by likelihood when
genotyping. default: 6.
--posterior-integration-limits N,M Integrate all genotype combinations in our posterior space
which include no more than N samples with their Mth best
data likelihood. default: 1,3.
--exclude-unobserved-genotypes Skip sample genotypings for which the sample has no supporting reads.
--genotype-variant-threshold N Limit posterior integration to samples where the second-best
genotype likelihood is no more than log(N) from the highest
genotype likelihood for the sample. default: ~unbounded
--use-mapping-quality Use mapping quality of alleles when calculating data likelihoods.
--harmonic-indel-quality Use a weighted sum of base qualities around an indel, scaled by the
distance from the indel. By default use a minimum BQ in flanking sequence.
--read-dependence-factor N Incorporate non-independence of reads by scaling successive
observations by this factor during data likelihood
calculations. default: 0.9
--genotype-qualities Calculate the marginal probability of genotypes and report as GQ in
each sample field in the VCF output.
Acknowledgments
The initial version of the wrapper was produced by Dan Blankenberg and upgraded by Anton Nekrutenko. TNG was developed by Bjoern Gruening.