*FreeBayes* uses short-read alignments
([BAM](http://samtools.sourceforge.net/SAMv1.pdf) files with
[Phred+33](http://en.wikipedia.org/wiki/Phred_quality_score) encoded quality
scores, now standard) for any number of individuals from a population and a
[reference genome](http://en.wikipedia.org/wiki/Reference_genome) (in
[FASTA](http://en.wikipedia.org/wiki/FASTA_format) 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](http://www.1000genomes.org/node/101))
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
sequencing](http://arxiv.org/abs/1207.3907) 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.
## Obtaining
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://github.com/ekg/freebayes.git
Note the use of --recursive. This is required in order to download all
nested git submodules for external repositories.
After you've already done the above to clone the most recent development
version, if you wish to compile a specific version of FreeBayes from, you
can then do something like the following:
git checkout v0.9.20 && git submodule update --recursive
### 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://github.com/ekg/freebayes.git
Cloning into 'freebayes'...
fatal: Unable to look up github.com (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.https://github.com/.insteadOf git://github.com/
## Compilation
FreeBayes requires g++ and the standard C and C++ development libraries.
Additionally, cmake is required for building the BamTools API.
make
Will build the executable freebayes, as well as the utilities bamfiltertech and
bamleftalign. These executables can be found in the `bin/` directory in the
repository.
Users may wish to install to e.g. /usr/local/bin (default), which is
accomplished via
sudo make install
## Usage
In its simplest operation, freebayes requires only two inputs: a FASTA reference
sequence, and a BAM-format alignment file sorted by reference position. For
instance:
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](http://github.com/pezmaster31/bamtools) 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
## Examples
Call variants assuming a diploid sample:
freebayes -f ref.fa aln.bam >var.vcf
Require at least 5 supporting observations to consider a variant:
freebayes -f ref.fa -C 5 aln.bam >var.vcf
Use a different ploidy:
freebayes -f ref.fa -p 4 aln.bam >var.vcf
Assume a pooled sample with a known number of genome copies. Note that this
means that each sample identified in the BAM file is assumed to have 32 genome
copies. When running with highh --ploidy settings, it may be required to set
`--use-best-n-alleles` to a low number to limit memory usage.
freebayes -f ref.fa -p 32 --use-best-n-alleles 4 --pooled-discrete aln.bam >var.vcf
Generate frequency-based calls for all variants passing input thresholds. You'd do
this in the case that you didn't know the number of samples in the pool.
freebayes -f ref.fa -F 0.01 -C 1 --pooled-continuous aln.bam >var.vcf
Use an input VCF (bgzipped + tabix indexed) to force calls at particular alleles:
freebayes -f ref.fa -@ in.vcf.gz aln.bam >var.vcf
Generate long haplotype calls over known variants:
freebayes -f ref.fa --haplotype-basis-alleles in.vcf.gz \
--haplotype-length 50 aln.bam
Naive variant calling: simply annotate observation counts of SNPs and indels:
freebayes -f ref.fa --haplotype-length 0 --min-alternate-count 1 \
--min-alternate-fraction 0 --pooled-continuous --report-monomorphic >var.vcf
Parallel operation (use 36 cores in this case):
freebayes-parallel <(fasta_generate_regions.py ref.fa.fai 100000) 36 \
-f ref.fa aln.bam >var.vcf
Note that any of the above examples can be made parallel by using the
scripts/freebayes-parallel script. If you find freebayes to be slow, you
should probably be running it in parallel using this script to run on a single
host, or generating a series of scripts, one per region, and run them on a
cluster.
## Calling variants: from fastq to VCF
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](http://bio-bwa.sourceforge.net/) or
[MOSAIK](https://github.com/wanpinglee/MOSAIK))
* 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](http://github.com/ekg/bamaddrg) to do so post-alignment.
* **Sort** the alignments (e.g. bamtools sort).
* **Mark duplicates**, for instance with [samtools
rmdup](http://samtools.sourceforge.net/) (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`
suitably.
* **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 emits a standard VCF 4.1 output 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
sample.
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](http://github.com/ekg/vcflib#vcffilter) in
[vcflib](http://github.com/ekg/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
characteristic](https://en.wikipedia.org/wiki/Receiver_operating_characteristic)
(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)
[bamaddrg](http://github.com/ekg/bamaddrg).
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.
## Performance tuning
If you find freebayes to be slow, or use large amounts of memory, consider the
following options:
- Set `--use-best-n-alleles 4`: this will reduce the number of alleles that are
considered, which will decrease runtime at the cost of sensitivity to
lower-frequency alleles at multiallelic loci. Calculating site qualities
requires O(samples\*genotypes) runtime, and the number of genotypes is
exponential in ploidy and the number of alleles that are considered, so this is
very important when working with high ploidy samples (and also
`--pooled-discrete`). By default, freebayes puts no limit on this.
- Remove `--genotype-qualities`: calculating genotype qualities requires
O(samples\*genotypes) memory.
- Set higher input thresholds. Require that N reads in one sample support an
allele in order to consider it: `--min-alternate-count N`, or that the allele
fraction in one sample is M: `--min-alternate-fraction M`. This will filter
noisy alleles. The defaults, `--min-alternate-count 2 --min-alternate-fraction
0.2`, are most-suitable for diploid, moderate-to-high depth samples, and should
be changed when working with different ploidy samples. Alternatively,
`--min-alternate-qsum` can be used to set a specific quality sum, which may be
more flexible than setting a hard count on the number of observations.
## 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
`--min-supporting-base-quality`.
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
`--min-alternate-fraction`
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
these*, because removing this information is very likely to reduce detection
power. To generate a report only including SNPs, use vcffilter post-call as
such:
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
haplotype.
### Effective base depth
By default, filters are left completely open.
Use `--experimental-gls` if you would like to integrate both base and mapping
quality are 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](http://www.hgsc.bcm.edu/software/snptools), which scales observation
quality by mapping quality. When `--experimental-gls` is given, *P(Obs|Genotype) ~
P(MappedCorrectly(Obs))P(SequencedCorrectly(Obs))*.
## 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.
bam_merger.sh | streaming_filter_or_process.sh | 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](http://github.com/ekg/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
bamfiltertech
[src/bamfiltertech.cpp](http://github.com/ekg/freebayes/blob/master/src/bamfilte
rtech.cpp), which could be used to filter out
technologies which have characteristic errors which may frustrate certain types
of variant detection.
## INDELs
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
inconsistency.
## 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](http://www.well.ox.ac.uk/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
philosophy](https://en.wikipedia.org/wiki/Unix_philosophy), 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
locus.
* **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
methods](http://bcbio.wordpress.com/2013/10/21/updated-comparison-of-variant-detection-methods-ensemble-freebayes-and-minimal-bam-preparation-pipelines/).
For a push-button solution to variant detection, from reads to variant calls,
look no further than the [gkno genome analysis platform](http://gkno.me/).
## Contributors
FreeBayes is made by:
- Erik Garrison
- Thomas Sibley
- Dillon Lee
- Patrick Marks
- Noah Spies
- Joshua Randall
- Jeremy Anderson
## Support
### email
Please report any issues or questions to the [freebayes mailing
list](https://groups.google.com/forum/#!forum/freebayes), [freebayes issue
tracker](https://github.com/ekg/freebayes/issues), or by email to