Overview:¶

To call variants from aligned short-read sequencing data, supply BAM files and a reference. FreeBayes will provide VCF output on standard out 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.

input¶

-b --bam FILE

Add FILE to the set of BAM files to be analyzed.

-L --bam-list FILE

A file containing a list of BAM files to be analyzed.

-c --stdin

Read BAM input on stdin.

-f --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.

-t --targets FILE

Limit analysis to targets listed in the BED-format FILE.

-r --region <chrom>:<start_position>-<end_position>

Limit analysis to the specified region, 0-base coordinates, end_position not included (same as BED format). Either '-' or '..' maybe used as a separator.

-s --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.

-A --cnv-map FILE

Read a copy number map from the BED file FILE, which has the format:

reference sequence, start, end, sample name, copy number

... for each region in each sample which does not have the default copy number as set by --ploidy.

output¶

-v --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.

-@ --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.

-l --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.

-P --pvar N

Report sites if the probability that there is a polymorphism at the site is greater than N. default: 0.0. Note that postfiltering is generally recommended over the use of this parameter.

population model¶

-T --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

-p --ploidy N

Sets the default ploidy for the analysis to N. default: 2

-J --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.

-K --pooled-continuous

Output all alleles which pass input filters, regardles of genotyping outcome or model.

reference allele¶

-Z --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¶

-I --no-snps

Ignore SNP alleles.

-i --no-indels

Ignore insertion and deletion alleles.

-X --no-mnps

Ignore multi-nuceotide polymorphisms, MNPs.

-u --no-complex Ignore complex events (composites of other classes).

-n --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)

-E --max-complex-gap N

--haplotype-length N

Allow haplotype calls with contiguous embedded matches of up to this length. (default: 3)

--min-repeat-size N

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. (default: 0, off)

--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¶

-O --dont-left-align-indels

Turn off left-alignment of indels, which is enabled by default.

input filters¶

-4 --use-duplicate-reads

Include duplicate-marked alignments in the analysis. default: exclude duplicates marked as such in alignments

-m --min-mapping-quality Q

Exclude alignments from analysis if they have a mapping quality less than Q. default: 1

-q --min-base-quality Q

Exclude alleles from analysis if their supporting base quality is less than Q. default: 0

-R --min-supporting-allele-qsum Q

Consider any allele in which the sum of qualities of supporting observations is at least Q. default: 0

-Y --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

-Q --mismatch-base-quality-threshold Q

Count mismatches toward --read-mismatch-limit if the base quality of the mismatch is >= Q. default: 10

-U --read-mismatch-limit N

Exclude reads with more than N mismatches where each mismatch has base quality >= mismatch-base-quality-threshold. default: ~unbounded

-z --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

-e --read-indel-limit N

Exclude reads with more than N separate gaps. default: ~unbounded

-0 --standard-filters

Use stringent input base and mapping quality filters

Equivalent to -m 30 -q 20 -R 0 -S 0

-F --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: 0.2

-C --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

-3 --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

-G --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

--max-coverage N

Do not process sites with greater than this coverage. default: no limit

population priors¶

-k --no-population-priors

Equivalent to --pooled-discrete --hwe-priors-off and removal of Ewens Sampling Formula component of priors.

mappability priors¶

-w --hwe-priors-off

Disable estimation of the probability of the combination arising under HWE given the allele frequency as estimated by observation frequency.

-V --binomial-obs-priors-off

Disable incorporation of prior expectations about observations. Uses read placement probability, strand balance probability, and read position (5'-3') probability.

-a --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.

-B --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.

-W --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.

-N --exclude-unobserved-genotypes

Skip sample genotypings for which the sample has no supporting reads.

-S --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

-j --use-mapping-quality

Use mapping quality of alleles when calculating data likelihoods.

-H --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.

-D --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.

debugging¶

-d --debug

Print debugging output.

-dd

Print more verbose debugging output (requires "make DEBUG")