NAME

samtools - Utilities for the Sequence Alignment/Map (SAM) format  

SYNOPSIS

samtools view -bt ref_list.txt -o aln.bam aln.sam.gz

samtools sort -T /tmp/aln.sorted -o aln.sorted.bam aln.bam

samtools index aln.sorted.bam

samtools idxstats aln.sorted.bam

samtools flagstat aln.sorted.bam

samtools stats aln.sorted.bam

samtools bedcov aln.sorted.bam

samtools depth aln.sorted.bam

samtools view aln.sorted.bam chr2:20,100,000-20,200,000

samtools merge out.bam in1.bam in2.bam in3.bam

samtools faidx ref.fasta

samtools fqidx ref.fastq

samtools tview aln.sorted.bam ref.fasta

samtools split merged.bam

samtools quickcheck in1.bam in2.cram

samtools dict -a GRCh38 -s "Homo sapiens" ref.fasta

samtools fixmate in.namesorted.sam out.bam

samtools mpileup -C50 -f ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam

samtools flags PAIRED,UNMAP,MUNMAP

samtools fastq input.bam > output.fastq

samtools fasta input.bam > output.fasta

samtools addreplacerg -r 'ID:fish' -r 'LB:1334' -r 'SM:alpha' -o output.bam input.bam

samtools collate -o aln.name_collated.bam aln.sorted.bam

samtools depad input.bam

samtools markdup in.algnsorted.bam out.bam

 

DESCRIPTION

Samtools is a set of utilities that manipulate alignments in the BAM format. It imports from and exports to the SAM (Sequence Alignment/Map) format, does sorting, merging and indexing, and allows to retrieve reads in any regions swiftly.

Samtools is designed to work on a stream. It regards an input file `-' as the standard input (stdin) and an output file `-' as the standard output (stdout). Several commands can thus be combined with Unix pipes. Samtools always output warning and error messages to the standard error output (stderr).

Samtools is also able to open a BAM (not SAM) file on a remote FTP or HTTP server if the BAM file name starts with `ftp://' or `http://'. Samtools checks the current working directory for the index file and will download the index upon absence. Samtools does not retrieve the entire alignment file unless it is asked to do so.

 

COMMANDS

Each command has its own man page which can be viewed using e.g. man samtools-view or with a recent GNU man using man samtools view. Below we have a brief summary of syntax and sub-command description.

Options common to all sub-commands are documented below in the GLOBAL COMMAND OPTIONS section.

view
samtools view [options] in.sam|in.bam|in.cram [region...]

With no options or regions specified, prints all alignments in the specified input alignment file (in SAM, BAM, or CRAM format) to standard output in SAM format (with no header by default).

You may specify one or more space-separated region specifications after the input filename to restrict output to only those alignments which overlap the specified region(s). Use of region specifications requires a coordinate-sorted and indexed input file.

Options exist to change the output format from SAM to BAM or CRAM, so this command also acts as a file format conversion utility.

sort
samtools sort [-l level] [-m maxMem] [-o out.bam] [-O format] [-n] [-t tag] [-T tmpprefix] [-@ threads] [in.sam|in.bam|in.cram]

Sort alignments by leftmost coordinates, or by read name when -n is used. An appropriate @HD-SO sort order header tag will be added or an existing one updated if necessary.

The sorted output is written to standard output by default, or to the specified file (out.bam) when -o is used. This command will also create temporary files tmpprefix.%d.bam as needed when the entire alignment data cannot fit into memory (as controlled via the -m option).

Consider using samtools collate instead if you need name collated data without a full lexicographical sort.

index
samtools index [-bc] [-m INT] aln.bam|aln.cram [out.index]

Index a coordinate-sorted BAM or CRAM file for fast random access. (Note that this does not work with SAM files even if they are bgzip compressed --- to index such files, use tabix(1) instead.)

This index is needed when region arguments are used to limit samtools view and similar commands to particular regions of interest.

If an output filename is given, the index file will be written to out.index. Otherwise, for a CRAM file aln.cram, index file aln.cram.crai will be created; for a BAM file aln.bam, either aln.bam.bai or aln.bam.csi will be created, depending on the index format selected.

idxstats
samtools idxstats in.sam|in.bam|in.cram

Retrieve and print stats in the index file corresponding to the input file. Before calling idxstats, the input BAM file should be indexed by samtools index.

If run on a SAM or CRAM file or an unindexed BAM file, this command will still produce the same summary statistics, but does so by reading through the entire file. This is far slower than using the BAM indices.

The output is TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads. It is written to stdout.

flagstat
samtools flagstat in.sam|in.bam|in.cram

Does a full pass through the input file to calculate and print statistics to stdout.

Provides counts for each of 13 categories based primarily on bit flags in the FLAG field. Each category in the output is broken down into QC pass and QC fail, which is presented as "#PASS + #FAIL" followed by a description of the category.

stats
samtools stats [options] in.sam|in.bam|in.cram [region...]

samtools stats collects statistics from BAM files and outputs in a text format. The output can be visualized graphically using plot-bamstats.

bedcov
samtools bedcov [options] region.bed in1.sam|in1.bam|in1.cram[...]

Reports the total read base count (i.e. the sum of per base read depths) for each genomic region specified in the supplied BED file. The regions are output as they appear in the BED file and are 0-based. Counts for each alignment file supplied are reported in separate columns.

depth
samtools depth [options] [in1.sam|in1.bam|in1.cram [in2.sam|in2.bam|in2.cram] [...]]

Computes the read depth at each position or region.

merge
samtools merge [-nur1f] [-h inh.sam] [-t tag] [-R reg] [-b list] out.bam in1.bam [in2.bam in3.bam ... inN.bam]

Merge multiple sorted alignment files, producing a single sorted output file that contains all the input records and maintains the existing sort order.

If -h is specified the @SQ headers of input files will be merged into the specified header, otherwise they will be merged into a composite header created from the input headers. If the @SQ headers differ in order this may require the output file to be re-sorted after merge.

The ordering of the records in the input files must match the usage of the -n and -t command-line options. If they do not, the output order will be undefined. See sort for information about record ordering.

faidx
samtools faidx <ref.fasta> [region1 [...]]

Index reference sequence in the FASTA format or extract subsequence from indexed reference sequence. If no region is specified, faidx will index the file and create <ref.fasta>.fai on the disk. If regions are specified, the subsequences will be retrieved and printed to stdout in the FASTA format.

The input file can be compressed in the BGZF format.

FASTQ files can be read and indexed by this command. Without using --fastq any extracted subsequence will be in FASTA format.

fqidx
samtools fqidx <ref.fastq> [region1 [...]]

Index reference sequence in the FASTQ format or extract subsequence from indexed reference sequence. If no region is specified, fqidx will index the file and create <ref.fastq>.fai on the disk. If regions are specified, the subsequences will be retrieved and printed to stdout in the FASTQ format.

The input file can be compressed in the BGZF format.

samtools fqidx should only be used on fastq files with a small number of entries. Trying to use it on a file containing millions of short sequencing reads will produce an index that is almost as big as the original file, and searches using the index will be very slow and use a lot of memory.

tview
samtools tview [-p chr:pos] [-s STR] [-d display] <in.sorted.bam> [ref.fasta]

Text alignment viewer (based on the ncurses library). In the viewer, press `?' for help and press `g' to check the alignment start from a region in the format like `chr10:10,000,000' or `=10,000,000' when viewing the same reference sequence.

split
samtools split [options] merged.sam|merged.bam|merged.cram

Splits a file by read group, producing one or more output files matching a common prefix (by default based on the input filename) each containing one read-group.

quickcheck
samtools quickcheck [options] in.sam|in.bam|in.cram [ ... ]

Quickly check that input files appear to be intact. Checks that beginning of the file contains a valid header (all formats) containing at least one target sequence and then seeks to the end of the file and checks that an end-of-file (EOF) is present and intact (BAM only).

Data in the middle of the file is not read since that would be much more time consuming, so please note that this command will not detect internal corruption, but is useful for testing that files are not truncated before performing more intensive tasks on them.

This command will exit with a non-zero exit code if any input files don't have a valid header or are missing an EOF block. Otherwise it will exit successfully (with a zero exit code).

dict
samtools dict ref.fasta|ref.fasta.gz

Create a sequence dictionary file from a fasta file.

fixmate
samtools fixmate [-rpcm] [-O format] in.nameSrt.bam out.bam

Fill in mate coordinates, ISIZE and mate related flags from a name-sorted alignment.

mpileup
samtools mpileup [-EB] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]

Generate textual pileup for one or multiple BAM files. For VCF and BCF output, please use the bcftools mpileup command instead. Alignment records are grouped by sample (SM) identifiers in @RG header lines. If sample identifiers are absent, each input file is regarded as one sample.

See the samtools-mpileup man page for a description of the pileup format and options.

flags
samtools flags INT|STR[,...]

Convert between textual and numeric flag representation.

FLAGS:

0x1PAIREDpaired-end (or multiple-segment) sequencing technology
0x2PROPER_PAIReach segment properly aligned according to the aligner
0x4UNMAPsegment unmapped
0x8MUNMAPnext segment in the template unmapped
0x10REVERSESEQ is reverse complemented
0x20MREVERSESEQ of the next segment in the template is reverse complemented
0x40READ1the first segment in the template
0x80READ2the last segment in the template
0x100SECONDARYsecondary alignment
0x200QCFAILnot passing quality controls
0x400DUPPCR or optical duplicate
0x800SUPPLEMENTARYsupplementary alignment

fastq/a
samtools fastq [options] in.bam
samtools fasta [options] in.bam

Converts a BAM or CRAM into either FASTQ or FASTA format depending on the command invoked. The files will be automatically compressed if the file names have a .gz or .bgzf extension.

The input to this program must be collated by name. Use samtools collate or samtools sort -n to ensure this.

collate
samtools collate [options] in.sam|in.bam|in.cram [<prefix>]

Shuffles and groups reads together by their names. A faster alternative to a full query name sort, collate ensures that reads of the same name are grouped together in contiguous groups, but doesn't make any guarantees about the order of read names between groups.

The output from this command should be suitable for any operation that requires all reads from the same template to be grouped together.

reheader
samtools reheader [-iP] in.header.sam in.bam

Replace the header in in.bam with the header in in.header.sam. This command is much faster than replacing the header with a BAM→SAM→BAM conversion.

By default this command outputs the BAM or CRAM file to standard output (stdout), but for CRAM format files it has the option to perform an in-place edit, both reading and writing to the same file. No validity checking is performed on the header, nor that it is suitable to use with the sequence data itself.

cat
samtools cat [-b list] [-h header.sam] [-o out.bam] in1.bam in2.bam [ ... ]

Concatenate BAMs or CRAMs. Although this works on either BAM or CRAM, all input files must be the same format as each other. The sequence dictionary of each input file must be identical, although this command does not check this. This command uses a similar trick to reheader which enables fast BAM concatenation.

rmdup
samtools rmdup [-sS] <input.srt.bam> <out.bam>

This command is obsolete. Use markdup instead.

addreplacerg
samtools addreplacerg [-r rg-line | -R rg-ID] [-m mode] [-l level] [-o out.bam] in.bam

Adds or replaces read group tags in a file.

calmd
samtools calmd [-Eeubr] [-C capQcoef] aln.bam ref.fasta

Generate the MD tag. If the MD tag is already present, this command will give a warning if the MD tag generated is different from the existing tag. Output SAM by default.

Calmd can also read and write CRAM files although in most cases it is pointless as CRAM recalculates MD and NM tags on the fly. The one exception to this case is where both input and output CRAM files have been / are being created with the no_ref option.

targetcut
samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] in.bam

This command identifies target regions by examining the continuity of read depth, computes haploid consensus sequences of targets and outputs a SAM with each sequence corresponding to a target. When option -f is in use, BAQ will be applied. This command is only designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].

phase
samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] in.bam

Call and phase heterozygous SNPs.

depad
samtools depad [-SsCu1] [-T ref.fa] [-o output] in.bam

Converts a BAM aligned against a padded reference to a BAM aligned against the depadded reference. The padded reference may contain verbatim "*" bases in it, but "*" bases are also counted in the reference numbering. This means that a sequence base-call aligned against a reference "*" is considered to be a cigar match ("M" or "X") operator (if the base-call is "A", "C", "G" or "T"). After depadding the reference "*" bases are deleted and such aligned sequence base-calls become insertions. Similarly transformations apply for deletions and padding cigar operations.

markdup
samtools markdup [-l length] [-r] [-s] [-T] [-S] in.algsort.bam out.bam

Mark duplicate alignments from a coordinate sorted file that has been run through samtools fixmate with the -m option. This program relies on the MC and ms tags that fixmate provides.

 

SAMTOOLS OPTIONS

These are options that are passed after the samtools command, before any sub-command is specified.

help--help
Display a brief usage message listing the samtools commands available. If the name of a command is also given, e.g., samtools help view, the detailed usage message for that particular command is displayed.

--version
Display the version numbers and copyright information for samtools and the important libraries used by samtools.

--version-only
Display the full samtools version number in a machine-readable format.

 

GLOBAL COMMAND OPTIONS

Several long-options are shared between multiple samtools sub-commands: --input-fmt, --input-fmt-option, --output-fmt, --output-fmt-option, and --reference. The input format is typically auto-detected so specifying the format is usually unnecessary and the option is included for completeness. Note that not all subcommands have all options. Consult the subcommand help for more details.

Format strings recognised are "sam", "bam" and "cram". They may be followed by a comma separated list of options as key or key=value. See below for examples.

The fmt-option arguments accept either a single option or option=value. Note that some options only work on some file formats and only on read or write streams. If value is unspecified for a boolean option, the value is assumed to be 1. The valid options are as follows.

level=INT
Output only. Specifies the compression level from 1 to 9, or 0 for uncompressed.
nthreads=INT
Specifies the number of threads to use during encoding and/or decoding. For BAM this will be encoding only. In CRAM the threads are dynamically shared between encoder and decoder.
reference=fasta_file
Specifies a FASTA reference file for use in CRAM encoding or decoding. It usually is not required for decoding except in the situation of the MD5 not being obtainable via the REF_PATH or REF_CACHE environment variables.
decode_md=0|1
CRAM input only; defaults to 1 (on). CRAM does not typically store MD and NM tags, preferring to generate them on the fly. When this option is 0 missing MD, NM tags will not be generated. It can be particularly useful when combined with a file encoded using store_md=1 and store_nm=1.
store_md=0|1
CRAM output only; defaults to 0 (off). CRAM normally only stores MD tags when the reference is unknown and lets the decoder generate these values on-the-fly (see decode_md).
store_nm=0|1
CRAM output only; defaults to 0 (off). CRAM normally only stores NM tags when the reference is unknown and lets the decoder generate these values on-the-fly (see decode_md).
ignore_md5=0|1
CRAM input only; defaults to 0 (off). When enabled, md5 checksum errors on the reference sequence and block checksum errors within CRAM are ignored. Use of this option is strongly discouraged.
required_fields=bit-field
CRAM input only; specifies which SAM columns need to be populated. By default all fields are used. Limiting the decode to specific columns can have significant performance gains. The bit-field is a numerical value constructed from the following table.
0x1SAM_QNAME
0x2SAM_FLAG
0x4SAM_RNAME
0x8SAM_POS
0x10SAM_MAPQ
0x20SAM_CIGAR
0x40SAM_RNEXT
0x80SAM_PNEXT
0x100SAM_TLEN
0x200SAM_SEQ
0x400SAM_QUAL
0x800SAM_AUX
0x1000SAM_RGAUX
name_prefix=string
CRAM input only; defaults to output filename. Any sequences with auto-generated read names will use string as the name prefix.
multi_seq_per_slice=0|1
CRAM output only; defaults to 0 (off). By default CRAM generates one container per reference sequence, except in the case of many small references (such as a fragmented assembly).
version=major.minor
CRAM output only. Specifies the CRAM version number. Acceptable values are "2.1" and "3.0".
seqs_per_slice=INT
CRAM output only; defaults to 10000.
slices_per_container=INT
CRAM output only; defaults to 1. The effect of having multiple slices per container is to share the compression header block between multiple slices. This is unlikely to have any significant impact unless the number of sequences per slice is reduced. (Together these two options control the granularity of random access.)
embed_ref=0|1
CRAM output only; defaults to 0 (off). If 1, this will store portions of the reference sequence in each slice, permitting decode without having requiring an external copy of the reference sequence.
no_ref=0|1
CRAM output only; defaults to 0 (off). If 1, sequences will be stored verbatim with no reference encoding. This can be useful if no reference is available for the file.
use_bzip2=0|1
CRAM output only; defaults to 0 (off). Permits use of bzip2 in CRAM block compression.
use_lzma=0|1
CRAM output only; defaults to 0 (off). Permits use of lzma in CRAM block compression.
lossy_names=0|1
CRAM output only; defaults to 0 (off). If 1, templates with all members within the same CRAM slice will have their read names removed. New names will be automatically generated during decoding. Also see the name_prefix option.

For example:

samtools view --input-fmt-option decode_md=0
    --output-fmt cram,version=3.0 --output-fmt-option embed_ref
    --output-fmt-option seqs_per_slice=2000 -o foo.cram foo.bam

 

REFERENCE SEQUENCES

The CRAM format requires use of a reference sequence for both reading and writing.

When reading a CRAM the @SQ headers are interrogated to identify the reference sequence MD5sum (M5: tag) and the local reference sequence filename (UR: tag). Note that http:// and ftp:// based URLs in the UR: field are not used, but local fasta filenames (with or without file://) can be used.

To create a CRAM the @SQ headers will also be read to identify the reference sequences, but M5: and UR: tags may not be present. In this case the -T and -t options of samtools view may be used to specify the fasta or fasta.fai filenames respectively (provided the .fasta.fai file is also backed up by a .fasta file).

The search order to obtain a reference is:

1.
Use any local file specified by the command line options (eg -T).
2.
Look for MD5 via REF_CACHE environment variable.
3.
Look for MD5 in each element of the REF_PATH environment variable.
4.
Look for a local file listed in the UR: header tag.

 

ENVIRONMENT VARIABLES

HTS_PATH
A colon-separated list of directories in which to search for HTSlib plugins. If $HTS_PATH starts or ends with a colon or contains a double colon (::), the built-in list of directories is searched at that point in the search.

If no HTS_PATH variable is defined, the built-in list of directories specified when HTSlib was built is used, which typically includes /usr/local/libexec/htslib and similar directories.

REF_PATH
A colon separated (semi-colon on Windows) list of locations in which to look for sequences identified by their MD5sums. This can be either a list of directories or URLs. Note that if a URL is included then the colon in http:// and ftp:// and the optional port number will be treated as part of the URL and not a PATH field separator. For URLs, the text %s will be replaced by the MD5sum being read.

If no REF_PATH has been specified it will default to http://www.ebi.ac.uk/ena/cram/md5/%s and if REF_CACHE is also unset, it will be set to $XDG_CACHE_HOME/hts-ref/%2s/%2s/%s. If $XDG_CACHE_HOME is unset, $HOME/.cache (or a local system temporary directory if no home directory is found) will be used similarly.

REF_CACHE
This can be defined to a single directory housing a local cache of references. Upon downloading a reference it will be stored in the location pointed to by REF_CACHE. When reading a reference it will be looked for in this directory before searching REF_PATH. To avoid many files being stored in the same directory, a pathname may be constructed using %nums and %s notation, consuming num characters of the MD5sum. For example /local/ref_cache/%2s/%2s/%s will create 2 nested subdirectories with the filenames in the deepest directory being the last 28 characters of the md5sum.

The REF_CACHE directory will be searched for before attempting to load via the REF_PATH search list. If no REF_PATH is defined, both REF_PATH and REF_CACHE will be automatically set (see above), but if REF_PATH is defined and REF_CACHE not then no local cache is used.

To aid population of the REF_CACHE directory a script misc/seq_cache_populate.pl is provided in the Samtools distribution. This takes a fasta file or a directory of fasta files and generates the MD5sum named files.

 

EXAMPLES

o
Import SAM to BAM when @SQ lines are present in the header:

samtools view -b aln.sam > aln.bam

If @SQ lines are absent:

samtools faidx ref.fa
samtools view -bt ref.fa.fai aln.sam > aln.bam

where ref.fa.fai is generated automatically by the faidx command.

o
Convert a BAM file to a CRAM file using a local reference sequence.

samtools view -C -T ref.fa aln.bam > aln.cram

 

LIMITATIONS

o
Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.

 

AUTHOR

Heng Li from the Sanger Institute wrote the original C version of samtools. Bob Handsaker from the Broad Institute implemented the BGZF library. Petr Danecek and Heng Li wrote the VCF/BCF implementation. James Bonfield from the Sanger Institute developed the CRAM implementation. Other large code contributions have been made by John Marshall, Rob Davies, Martin Pollard, Andrew Whitwham, Valeriu Ohan (all while primarily at the Sanger Institute), with numerous other smaller but valuable contributions. See the per-command manual pages for further authorship.

 

SEE ALSO

samtools-addreplacerg(1), samtools-bedcov(1), samtools-calmd(1), samtools-cat(1), samtools-collate(1), samtools-depad(1), samtools-depth(1), samtools-dict(1), samtools-faidx(1), samtools-fasta(1), samtools-fastq(1), samtools-fixmate(1), samtools-flags(1), samtools-flagstat(1), samtools-fqidx(1), samtools-idxstats(1), samtools-index(1), samtools-markdup(1), samtools-merge(1), samtools-mpileup(1), samtools-phase(1), samtools-quickcheck(1), samtools-reheader(1), samtools-rmdup(1), samtools-sort(1), samtools-split(1), samtools-stats(1), samtools-targetcut(1), samtools-tview(1), samtools-view(1), bcftools(1), sam(5), tabix(1)

Samtools website: <http://www.htslib.org/>
File format specification of SAM/BAM,CRAM,VCF/BCF: <http://samtools.github.io/hts-specs>
Samtools latest source: <https://github.com/samtools/samtools>
HTSlib latest source: <https://github.com/samtools/htslib>
Bcftools website: <http://samtools.github.io/bcftools>