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Short Description
NGSeasy Freebayes
Full Description

NGSeasy Freebayes

freebayes, a haplotype-based variant detector


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

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

<img src="" width=500/>

FreeBayes uses short-read alignments
(BAM files with
Phred+33 encoded quality
scores, now standard) for any number of individuals from a population and a
reference genome (in
FASTA format)
to determine the most-likely combination of genotypes for the population at
each position in the reference. It reports positions which it finds putatively
polymorphic in variant call file (VCF)
format. It can also use an input set of variants (VCF) as a source of prior
information, and a copy number variant map (BED) to define non-uniform ploidy
variation across the samples under analysis.

Citing freebayes

A preprint Haplotype-based variant detection from short-read
provides an overview of the
statistical models
used in FreeBayes. We ask that you cite this paper if you use FreeBayes in
work that leads to publication.

Please use this citation format:

Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing.
arXiv preprint arXiv:1207.3907 [q-bio.GN] 2012

If possible, please also refer to the version number provided by freebayes when
it is run without arguments or with the --help option. For example, you
should see something like this:

version:  v0.9.10-3-g47a713e-dirty

This provides both a point release number and a git commit id, which will
ensure precise reproducibility of results.


To download FreeBayes, please use git to download the most recent development
tree. Currently, the tree is hosted on github, and can be obtained via:

git clone --recursive git://

Note the use of --recursive. This is required, as the project contains some
nested git submodules for external repositories.

Resolving proxy issues with git

Depending on your local network configuration, you may have problems obtaining
freebayes via git. If you see something like this you may be behind a proxy
that blocks access to standard git:// port (9418).

$ git clone --recursive git://
Cloning into 'freebayes'...
fatal: Unable to look up (port 9418) (Name or service not known)

Luckily, if you have access to https:// on port 443, then you can use this
'magic' command as a workaround to enable download of the submodules:

git config --global url. git://


FreeBayes requires g++ and the standard C and C++ development libraries.
Additionally, cmake is required for building the BamTools API.


Will build the executable freebayes, as well as the utilities bamfiltertech and
bamleftalign. These executables can be found in the bin/ directory in the

Users may wish to install to e.g. /usr/local/bin (default), which is
accomplished via

sudo make install


In its simplest operation, freebayes requires only two inputs: a FASTA reference
sequence, and a BAM-format alignment file sorted by reference position. For

freebayes --fasta-reference h.sapiens.fasta NA20504.bam

... produce (on standard output) a VCF file on standard out describing
all SNPs, INDELs, MNPs, and Complex events between the reference and the
alignments in NA20504.bam. In order to produce correct output, the reference
supplied must be the reference to which NA20504.bam was aligned.

Users may specify any number of BAM files on the command line. FreeBayes uses
the BamTools API to open and parse
these files in parallel, virtually merging them at runtime into one logical
file with a merged header.

For a description of available command-line options and their defaults, run:

freebayes --help

Calling variants

You've sequenced some samples. You have a reference genome or assembled set of
contigs, and you'd like to determine reference-relative variants in your
samples. You can use freebayes to detect the variants, following these steps:

  • Align your reads to a suitable reference (e.g. with
    bwa or
  • Ensure your alignments have read groups attached so their sample may be
    identified by freebayes. Aligners allow you to do this, but you can also use
    bamaddrg to do so post-alignment.
  • Sort the alignments (e.g. bamtools sort).
  • Mark duplicates, for instance with samtools
    (if PCR was used in the preparation of
    your sequencing library)
  • Run freebayes on all your alignment data simultaneously, generating a
    VCF. The default settings should work for most use cases, but if your samples
    are not diploid, set the --ploidy and adjust the --min-alternate-fraction
  • Filter the output e.g. using reported QUAL and/or depth (DP) or
    observation count (AO).
  • Interpret your results.
  • (possibly, Iterate the variant detection process in response to insight
    gained from your interpretation)

FreeBayes a standard VCF 4.1 outut stream. This format is designed for the
probabilistic description of allelic variants within a population of samples,
but it is equally suited to describing the probability of variation in a single

Of primary interest to most users is the QUAL field, which estimates the
probability that there is a polymorphism at the loci described by the record.
In freebayes, this value can be understood as 1 - P(locus is homozygous given
the data). It is recommended that users use this value to filter their
results, rather than accepting anything output by freebayes as ground truth.

By default, records are output even if they have very low probability of
variation, in expectation that the VCF will be filtered using tools such as
vcffilter in
vcflib, which is also included in the
repository under vcflib/. For instance,

freebayes -f ref.fa aln.bam | vcffilter -f "QUAL > 20" >results.vcf

removes any sites with estimated probability of not being polymorphic less than
phred 20 (aka 0.01), or probability of polymorphism > 0.99.

In simulation, the receiver-operator

(ROC) tends to have a very sharp inflection between Q1 and Q30, depending on
input data characteristics, and a filter setting in this range should provide
decent performance. Users are encouraged to examine their output and both
variants which are retained and those they filter out. Most problems tend to
occur in low-depth areas, and so users may wish to remove these as well, which
can also be done by filtering on the DP flag.

Calling variants in a population

FreeBayes is designed to be run on many individuals from the same population
(e.g. many human individuals) simultaneously. The algorithm exploits a neutral
model of allele diffusion to impute most-confident genotypings
across the entire population. In practice, the discriminant power of the
method will improve if you run multiple samples simultaneously. In other
words, if your
study has multiple individuals, you should run freebayes against them at the
same time. This also ensures consistent reporting of information about
evidence for all samples at any locus where any are apparently polymorphic.

To call variants in a population of samples, each alignment must have a read
group identifier attached to it (RG tag), and the header of the BAM file in
which it resides must map the RG tags to sample names (SM). Furthermore, read
group IDs must be unique across all the files used in the analysis. One read
group cannot map to multiple samples. The reason this is required is that
freebayes operates on a virtually merged BAM stream provided by the BamTools
API. If merging the files in your analysis using bamtools merge would generate
a file in which multiple samples map to the same RG, the files are not suitable
for use in population calling, and they must be modified.

Users may add RG tags to BAM files which were generated without this
information by using (as mentioned in "Calling variants" above)
If you have many files corresponding to
many individuals, add a unique read group and sample name to each, and then
open them all simultaneously with freebayes. The VCF output will have one
column per sample in the input.

Observation filters and qualities

Input filters

FreeBayes filters its input so as to ignore low-confidence alignments and
alleles which are only supported by low-quality sequencing observations (see
--min-mapping-quality and --min-base-quality). It also will only evaluate a
position if at least one read has mapping quality of
--min-supporting-mapping-quality and one allele has quality of at least

Reads with more than a fixed number of high-quality mismatches can be excluded
by specifying --read-mismatch-limit. This is meant as a workaround when
mapping quality estimates are not appropriately calibrated.

Reads marked as duplicates in the BAM file are ignored, but this can be
disabled for testing purposes by providing --use-duplicate-reads. FreeBayes
does not mark duplicates on its own, you must use another process to do this.

Observation thresholds

As a guard against spurious variation caused by sequencing artifacts, positions
are skipped when no more than --min-alternate-count or
non-clonal observations of an alternate are found in one sample. These default
to 2 and 0.2 respectively. The default setting of --min-alternate-fraction 0.2 is suitable for diploid samples but should be changed for ploidy > 2.

Allele type exclusion

FreeBayes provides a few methods to ignore certain classes of allele, e.g.
--no-indels and --no-mnps. Users are strongly cautioned against using
, because removing this information is very likely to reduce detection
power. To generate a report only including SNPs, use vcffilter post-call as

freebayes ... | vcffilter -f "TYPE = snp"

Observation qualities

FreeBayes estimates observation quality using several simple heuristics based
on manipulations of the phred-scaled base qualities:

  • For single-base observations, mismatches and reference observations: the
    un-adjusted base quality provided in the BAM alignment record.
  • For insertions: the mean quality of the bases inside of the putatively
    inserted sequence.
  • For deletions: the mean quality of the bases flanking the putatively
    deleted sequence.
  • For haplotypes: the mean quality of allele observations within the

Effective base depth

By default, filters are left completely open, as both mapping quality and base
quality are incorporated into the reported site quality (QUAL in the VCF) and
genotype quality (GQ, when supplying --genotype-qualities). This integration
is driven by the "Effective Base Depth" metric first developed in
snpTools, which scales observation
quality by mapping quality. In short, P(Obs|Genotype) ~

Stream processing

FreeBayes can read BAM from standard input --stdin instead of directly from
files. This allows the application of any number of streaming BAM filters and
calibrators to its input. | | freebayes --stdin ...

This pattern allows the adjustment of alignments without rewriting BAM files,
which could be expensive depending on context and available storage. A prime
example of this would be graph-based realignment of reads to known variants as
implemented in glia.

Using this pattern, you can filter out reads with certain criteria using
bamtools filter without having to modify the input BAM file. You can also use
the bamtools API to write your own custom filters in C++. An example filter is
src/bamfiltertech.cpp, which could be used to filter out
technologies which have characteristic errors which may frustrate certain types
of variant detection.


In principle, any gapped aligner which is sensitive to indels will
produce satisfactory input for use by freebayes. Due to potential ambiguity,
indels are
not parsed when they overlap the beginning or end of alignment boundaries.

When calling indels, it is important to homogenize the positional distribution
of insertions and deletions in the input by using left realignment. This is
now done automatically by freebayes, but the behavior can be turned off via
--dont-left-align-indels flag. You probably don't want to do this.

Left realignment will place all indels in homopolymer and microsatellite
repeats at the same position, provided that doing so does not introduce
mismatches between the read and reference other than the indel. This method
computationally inexpensive and handles the most common classes of alignment

Haplotype calls

As freebayes is haplotype-based, left-alignment is necessary only for the
determination of candidate polymorphic loci. Once such loci are determined,
haplotype observations are extracted from reads where:

  1. putative variants lie within --haplotype-window bases of each other
    (default 3bp),
  2. the reference sequence has repeats (e.g. microsatellites or STRs are called
    as one haplotype),
  3. the haplotype which is called has Shannon entropy less than
    --min-repeat-entropy, which is off by default but can be set to ~1 for
    optimal genotyping of indels in lower-complexity sequence.

After a haplotype window is determined by greedily expanding the window across
overlapping haplotype observations, all reads overlapping the window are used
to establish data likelihoods, P(Observations|Genotype), for all haplotypes
which have sufficient support to pass the input filters.

Partial observations are considered to support those haplotypes which they
could match exactly. For expedience, only haplotypes which are contiguously
observed by the reads are considered as putative alleles in this process. This
differs from other haplotype-based methods, such as
Platypus, which consider all possible
haplotypes composed of observed component alleles (SNPs, indels) in a given
region when generating likelihoods.

The primary adantages of this approach are conceptual simplicity and
performance, and it is primarily limited in the case of short reads, an issue
that is mitigated by increasing read lengths. Also, a hybrid approach must be
used to call haplotypes from high-error rate long reads.

Re-genotyping known variants and calling long haplotypes

For longer reads with higher error rates, it is possible to generate long
haplotypes in two passes over the data. For instance, if we had very long
reads (e.g. >10kb) at moderate depth and high error rate (>5%) such as might be
produced by PacBio, we could do something like:

freebayes -f ref.fa aln.bam | vcffilter -f "QUAL > 20" >vars.vcf

... thus generating candidate variants of suitable quality using the default
detection window. We can then use these as "basis alleles" for the observation
of haplotypes, considering all other putative variants supported by the
alignment to be sequencing errors:

freebayes -f ref.fa --haplotype-window 500 \
    --haplotype-basis-alleles vars.vcf aln.bam >haps.vcf

These steps should allow us to read long haplotypes directly from input data
with high error rates.

The high error rate means that beyond a small window each read will contain a
completely different literal haplotype. To a point, this property improves our
signal to noise ratio and can effectively filter out sequencing errors at the
point of the input filters, but it also decreases the effective observation
depth will prevent the generation of any calls if a long --haplotype-window
is combined with high a sequencing error rate.

Best practices and design philosophy

FreeBayes follows the patterns suggested by the Unix
, which promotes the
development of simple, modular systems that perform a single function, and can
be combined into more complex systems using stream processing of common
interchange formats.

FreeBayes incorporates a number of features in order to reduce the complexity
of variant detection for researchers and developers:

  • Indel realignment is accomplished internally using a read-independent
    method, and issues resulting from discordant alignments are dramatically
    reducedy through the direct detection of haplotypes.
  • The need for base quality recalibration is avoided through the direct
    detection of haplotypes. Sequencing platform errors tend to cluster (e.g. at
    the ends of reads), and generate unique, non-repeating haplotypes at a given
  • Variant quality recalibration is avoided by incorporating a number of
    metrics, such as read placement bias and allele balance, directly into the
    Bayesian model. (Our upcoming publication will discuss this in more detail.)

A minimal pre-processing pipeline similar to that described in "Calling
variants" should be sufficient for most uses. For more information, please
refer to a recent post by Brad Chapman on minimal BAM preprocessing

For a push-button solution to variant detection, from reads to variant calls,
look no further than the gkno genome analysis platform.


Please report any issues or questions to the freebayes mailing
, freebayes issue
, or by email to

Note that if you encounter issues with the development HEAD and you would like
a quick workaround for an issue that is likely to have been reintroduced
recently, you can use git checkout to step back a few revisions.

git checkout [git-commit-id]

It will also help with debugging to know if a problem has arisen in recent

Docker Pull Command