samtools(1) Bioinformatics tools samtools(1)NAMEsamtools - Utilities for the Sequence Alignment/Map (SAM) format
SYNOPSISsamtools 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 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 -gf 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 out‐
put.bam input.bam
samtools collate aln.sorted.bam aln.name_collated.bam
samtools depad input.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 AND OPTIONS
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).
You may specify one or more space-separated region specifica‐
tions 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 (in BAM or CRAM format).
The -b, -C, -1, -u, -h, -H, and -c options change the output
format from the default of headerless SAM, and the -o and -U
options set the output file name(s).
The -t and -T options provide additional reference data. One
of these two options is required when SAM input does not con‐
tain @SQ headers, and the -T option is required whenever
writing CRAM output.
The -L, -r, -R, -q, -l, -m, -f, and -F options filter the
alignments that will be included in the output to only those
alignments that match certain criteria.
The -x, -B, and -s options modify the data which is contained
in each alignment.
Finally, the -@ option can be used to allocate additional
threads to be used for compression, and the -? option
requests a long help message.
REGIONS:
Regions can be specified as: RNAME[:STARTPOS[-ENDPOS]] and
all position coordinates are 1-based.
Important note: when multiple regions are given, some align‐
ments may be output multiple times if they overlap more than
one of the specified regions.
Examples of region specifications:
chr1 Output all alignments mapped to the reference
sequence named `chr1' (i.e. @SQ SN:chr1).
chr2:1000000
The region on chr2 beginning at base position
1,000,000 and ending at the end of the chromosome.
chr3:1000-2000
The 1001bp region on chr3 beginning at base posi‐
tion 1,000 and ending at base position 2,000
(including both end positions).
'*' Output the unmapped reads at the end of the file.
(This does not include any unmapped reads placed on
a reference sequence alongside their mapped mates.)
. Output all alignments. (Mostly unnecessary as not
specifying a region at all has the same effect.)
OPTIONS:
-b Output in the BAM format.
-C Output in the CRAM format (requires -T).
-1 Enable fast BAM compression (implies -b).
-u Output uncompressed BAM. This option saves time
spent on compression/decompression and is thus pre‐
ferred when the output is piped to another samtools
command.
-h Include the header in the output.
-H Output the header only.
-c Instead of printing the alignments, only count them
and print the total number. All filter options,
such as -f, -F, and -q, are taken into account.
-? Output long help and exit immediately.
-o FILE Output to FILE [stdout].
-U FILE Write alignments that are not selected by the vari‐
ous filter options to FILE. When this option is
used, all alignments (or all alignments intersect‐
ing the regions specified) are written to either
the output file or this file, but never both.
-t FILE A tab-delimited FILE. Each line must contain the
reference name in the first column and the length
of the reference in the second column, with one
line for each distinct reference. Any additional
fields beyond the second column are ignored. This
file also defines the order of the reference
sequences in sorting. If you run: `samtools faidx
<ref.fa>', the resulting index file <ref.fa>.fai
can be used as this FILE.
-T FILE A FASTA format reference FILE, optionally com‐
pressed by bgzip and ideally indexed by samtools
faidx. If an index is not present, one will be
generated for you.
-L FILE Only output alignments overlapping the input BED
FILE [null].
-r STR Only output alignments in read group STR [null].
-R FILE Output alignments in read groups listed in FILE
[null].
-q INT Skip alignments with MAPQ smaller than INT [0].
-l STR Only output alignments in library STR [null].
-m INT Only output alignments with number of CIGAR bases
consuming query sequence ≥ INT [0]
-f INT Only output alignments with all bits set in INT
present in the FLAG field. INT can be specified in
hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/) or
in octal by beginning with `0' (i.e. /^0[0-7]+/)
[0].
-F INT Do not output alignments with any bits set in INT
present in the FLAG field. INT can be specified in
hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/) or
in octal by beginning with `0' (i.e. /^0[0-7]+/)
[0].
-x STR Read tag to exclude from output (repeatable) [null]
-B Collapse the backward CIGAR operation.
-s FLOAT Integer part is used to seed the random number gen‐
erator [0]. Part after the decimal point sets the
fraction of templates/pairs to subsample [no sub‐
sampling].
-@ INT Number of BAM compression threads to use in addi‐
tion to main thread [0].
-S Ignored for compatibility with previous samtools
versions. Previously this option was required if
input was in SAM format, but now the correct format
is automatically detected by examining the first
few characters of input.
sort samtools sort [-l level] [-m maxMem] [-o out.bam] [-O format]
[-n] [-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).
Options:
-l INT Set the desired compression level for the final
output file, ranging from 0 (uncompressed) or 1
(fastest but minimal compression) to 9 (best com‐
pression but slowest to write), similarly to
gzip(1)'s compression level setting.
If -l is not used, the default compression level
will apply.
-m INT Approximately the maximum required memory per
thread, specified either in bytes or with a K, M,
or G suffix. [768 MiB]
-n Sort by read names (i.e., the QNAME field) rather
than by chromosomal coordinates.
-o FILE Write the final sorted output to FILE, rather than
to standard output.
-O FORMAT Write the final output as sam, bam, or cram.
By default, samtools tries to select a format
based on the -o filename extension; if output is
to standard output or no format can be deduced,
bam is selected.
-T PREFIX Write temporary files to PREFIX.nnnn.bam. By
default, any temporary files are written alongside
the input file, as in.bam.tmp.nnnn.bam.
-@ INT Set number of sorting and compression threads. By
default, operation is single-threaded.
Historically samtools sort also accepted a less flexible way
of specifying the final and temporary output filenames:
samtools sort [-f] [-o] in.bam out.prefix
This has now been removed. The previous out.prefix argument
(and -f option, if any) should be changed to an appropriate
combination of -T PREFIX and -o FILE. The previous -o option
should be removed, as output defaults to standard output.
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 writ‐
ten 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.
Options:
-b Create a BAI index. This is currently the default
when no format options are used.
-c Create a CSI index. By default, the minimum interval
size for the index is 2^14, which is the same as the
fixed value used by the BAI format.
-m INT Create a CSI index, with a minimum interval size of
2^INT.
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
must be indexed by samtools index.
The output is TAB-delimited with each line consisting of ref‐
erence 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.
The first row of output gives the total number of reads that
are QC pass and fail (according to flag bit 0x200). For exam‐
ple:
122 + 28 in total (QC-passed reads + QC-failed reads)
Which would indicate that there are a total of 150 reads in
the input file, 122 of which are marked as QC pass and 28 of
which are marked as "not passing quality controls"
Following this, additional categories are given for reads
which are:
secondary
0x100 bit set
supplementary
0x800 bit set
duplicates
0x400 bit set
mapped 0x4 bit not set
paired in sequencing
0x1 bit set
read1 both 0x1 and 0x40 bits set
read2 both 0x1 and 0x80 bits set
properly paired
both 0x1 and 0x2 bits set and 0x4 bit not set
with itself and mate mapped
0x1 bit set and neither 0x4 nor 0x8 bits set
singletons
both 0x1 and 0x8 bits set and bit 0x4 not set
And finally, two rows are given that additionally filter on
the reference name (RNAME), mate reference name (MRNM), and
mapping quality (MAPQ) fields:
with mate mapped to a different chr
0x1 bit set and neither 0x4 nor 0x8 bits set
and MRNM not equal to RNAME
with mate mapped to a different chr (mapQ>=5)
0x1 bit set and neither 0x4 nor 0x8 bits set
and MRNM not equal to RNAME and MAPQ >= 5
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.
Options:
-c, --coverage MIN,MAX,STEP
Set coverage distribution to the specified range
(MIN, MAX, STEP all given as integers) [1,1000,1]
-d, --remove-dups
Exclude from statistics reads marked as duplicates
-f, --required-flag STR|INT
Required flag, 0 for unset. See also `samtools flags`
[0]
-F, --filtering-flag STR|INT
Filtering flag, 0 for unset. See also `samtools
flags` [0]
--GC-depth FLOAT
the size of GC-depth bins (decreasing bin size
increases memory requirement) [2e4]
-h, --help
This help message
-i, --insert-size INT
Maximum insert size [8000]
-I, --id STR
Include only listed read group or sample name []
-l, --read-length INT
Include in the statistics only reads with the given
read length []
-m, --most-inserts FLOAT
Report only the main part of inserts [0.99]
-P, --split-prefix STR
A path or string prefix to prepend to filenames out‐
put when creating categorised statistics files with
-S/--split. [input filename]
-q, --trim-quality INT
The BWA trimming parameter [0]
-r, --ref-seq FILE
Reference sequence (required for GC-depth and mis‐
matches-per-cycle calculation). []
-S, --split TAG
In addition to the complete statistics, also output
categorised statistics based on the tagged field TAG
(e.g., use --split RG to split into read groups).
Categorised statistics are written to files named
<prefix>_<value>.bamstat, where prefix is as given by
--split-prefix (or the input filename by default) and
value has been encountered as the specified tagged
field's value in one or more alignment records.
-t, --target-regions FILE
Do stats in these regions only. Tab-delimited file
chr,from,to, 1-based, inclusive. []
-x, --sparse
Suppress outputting IS rows where there are no inser‐
tions.
bedcov samtools bedcov region.bed in1.sam|in1.bam|in1.cram[...]
Reports read depth per genomic region, as specified in the
supplied BED file.
depth samtools depth [options] [in1.sam|in1.bam|in1.cram
[in2.sam|in2.bam|in2.cram] [...]]
Computes the depth at each position or region.
Options:
-a Output all positions (including those with zero
depth)
-a -a, -aa
Output absolutely all positions, including unused
reference sequences
-b FILE Compute depth at list of positions or regions in
specified BED FILE. []
-f FILE Use the BAM files specified in the FILE (a file of
filenames, one file per line) []
-l INT Ignore reads shorter than INT
-m, -d INT
Truncate reported depth at a maximum of INT reads.
[8000]
-q INT Only count reads with base quality greater than INT
-Q INT Only count reads with mapping quality greater than
INT
-r CHR:FROM-TO
Only report depth in specified region.
merge samtools merge [-nur1f] [-h inh.sam] [-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 head‐
ers. If in the process of merging @SQ lines for coordinate
sorted input files, a conflict arises as to the order (for
example input1.bam has @SQ for a,b,c and input2.bam has
b,a,c) then the resulting output file will need to be re-
sorted back into coordinate order.
Unless the -c or -p flags are specified then when merging @RG
and @PG records into the output header then any IDs found to
be duplicates of existing IDs in the output header will have
a suffix appended to them to diffientiate them from similar
header records from other files and the read records will be
updated to reflect this.
OPTIONS:
-1 Use zlib compression level 1 to compress the output.
-b FILE List of input BAM files, one file per line.
-f Force to overwrite the output file if present.
-h FILE Use the lines of FILE as `@' headers to be copied to
out.bam, replacing any header lines that would other‐
wise be copied from in1.bam. (FILE is actually in
SAM format, though any alignment records it may con‐
tain are ignored.)
-n The input alignments are sorted by read names rather
than by chromosomal coordinates
-R STR Merge files in the specified region indicated by STR
[null]
-r Attach an RG tag to each alignment. The tag value is
inferred from file names.
-u Uncompressed BAM output
-c When several input files contain @RG headers with the
same ID, emit only one of them (namely, the header
line from the first file we find that ID in) to the
merged output file. Combining these similar headers
is usually the right thing to do when the files being
merged originated from the same file.
Without -c, all @RG headers appear in the output
file, with random suffices added to their IDs where
necessary to differentiate them.
-p Similarly, for each @PG ID in the set of files to
merge, use the @PG line of the first file we find
that ID in rather than adding a suffix to differenti‐
ate similar IDs.
faidx samtools faidx <ref.fasta> [region1 [...]]
Index reference sequence in the FASTA format or extract sub‐
sequence 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.
The sequences in the input file should all have different
names. If they do not, indexing will emit a warning about
duplicate sequences and retrieval will only produce subse‐
quences from the first sequence with the duplicated name.
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 align‐
ment start from a region in the format like
`chr10:10,000,000' or `=10,000,000' when viewing the same
reference sequence.
Options:
-d display Output as (H)tml or (C)urses or (T)ext
-p chr:pos Go directly to this position
-s STR Display only alignments from this sample or
read group
split samtools split [options] merged.sam|merged.bam|merged.cram
Splits a file by read group.
Options:
-u FILE1 Put reads with no RG tag or an unrecognised RG
tag into FILE1
-u FILE1:FILE2
As above, but assigns an RG tag as given in the
header of FILE2
-f STRING Output filename format string (see below)
["%*_%#.%."]
-v Verbose output
Format string expansions:
%% %
%* basename
%# @RG index
%! @RG ID
%. output format filename extension
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 for‐
mats) 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 test‐
ing 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).
Options:
-v Verbose output: will additionally print the names of
all input files that don't pass the check to stdout.
Multiple -v options will cause additional messages
regarding check results to be printed to stderr.
dict samtools dict <ref.fasta|ref.fasta.gz>
Create a sequence dictionary file from a fasta file.
OPTIONS:
-a, --assembly STR
Specify the assembly for the AS tag.
-H, --no-header
Do not print the @HD header line.
-o, --output FILE
Output to FILE [stdout].
-s, --species STR
Specify the species for the SP tag.
-u, --uri STR
Specify the URI for the UR tag. Defaults to the
absolute path of ref.fasta unless reading from
stdin.
fixmate samtools fixmate [-rpc] [-O format] in.nameSrt.bam out.bam
Fill in mate coordinates, ISIZE and mate related flags from a
name-sorted alignment.
OPTIONS:
-r Remove secondary and unmapped reads.
-p Disable FR proper pair check.
-c Add template cigar ct tag.
-O FORMAT Write the final output as sam, bam, or cram.
By default, samtools tries to select a format
based on the output filename extension; if output
is to standard output or no format can be deduced,
bam is selected.
mpileup samtools mpileup [-EBugp] [-C capQcoef] [-r reg] [-f in.fa]
[-l list] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]
Generate VCF, BCF or pileup for one or multiple BAM files.
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.
In the pileup format (without -u or -g), each line represents
a genomic position, consisting of chromosome name, 1-based
coordinate, reference base, the number of reads covering the
site, read bases, base qualities and alignment mapping quali‐
ties. Information on match, mismatch, indel, strand, mapping
quality and start and end of a read are all encoded at the
read base column. At this column, a dot stands for a match to
the reference base on the forward strand, a comma for a match
on the reverse strand, a '>' or '<' for a reference skip,
`ACGTN' for a mismatch on the forward strand and `acgtn' for
a mismatch on the reverse strand. A pattern `\+[0-9]+[ACGT‐
Nacgtn]+' indicates there is an insertion between this refer‐
ence position and the next reference position. The length of
the insertion is given by the integer in the pattern, fol‐
lowed by the inserted sequence. Similarly, a pattern
`-[0-9]+[ACGTNacgtn]+' represents a deletion from the refer‐
ence. The deleted bases will be presented as `*' in the fol‐
lowing lines. Also at the read base column, a symbol `^'
marks the start of a read. The ASCII of the character follow‐
ing `^' minus 33 gives the mapping quality. A symbol `$'
marks the end of a read segment.
Input Options:
-6, --illumina1.3+
Assume the quality is in the Illumina 1.3+ encod‐
ing.
-A, --count-orphans
Do not skip anomalous read pairs in variant call‐
ing.
-b, --bam-list FILE
List of input BAM files, one file per line [null]
-B, --no-BAQ
Disable probabilistic realignment for the computa‐
tion of base alignment quality (BAQ). BAQ is the
Phred-scaled probability of a read base being mis‐
aligned. Applying this option greatly helps to
reduce false SNPs caused by misalignments.
-C, --adjust-MQ INT
Coefficient for downgrading mapping quality for
reads containing excessive mismatches. Given a read
with a phred-scaled probability q of being gener‐
ated from the mapped position, the new mapping
quality is about sqrt((INT-q)/INT)*INT. A zero
value disables this functionality; if enabled, the
recommended value for BWA is 50. [0]
-d, --max-depth INT
At a position, read maximally INT reads per input
BAM. [250]
-E, --redo-BAQ
Recalculate BAQ on the fly, ignore existing BQ tags
-f, --fasta-ref FILE
The faidx-indexed reference file in the FASTA for‐
mat. The file can be optionally compressed by
bgzip. [null]
-G, --exclude-RG FILE
Exclude reads from readgroups listed in FILE (one
@RG-ID per line)
-l, --positions FILE
BED or position list file containing a list of
regions or sites where pileup or BCF should be gen‐
erated. If BED, positions are 0-based half-open
[null]
-q, -min-MQ INT
Minimum mapping quality for an alignment to be used
[0]
-Q, --min-BQ INT
Minimum base quality for a base to be considered
[13]
-r, --region STR
Only generate pileup in region. Requires the BAM
files to be indexed. If used in conjunction with
-l then considers the intersection of the two
requests. STR [all sites]
-R, --ignore-RG
Ignore RG tags. Treat all reads in one BAM as one
sample.
--rf, --incl-flags STR|INT
Required flags: skip reads with mask bits unset
[null]
--ff, --excl-flags STR|INT
Filter flags: skip reads with mask bits set
[UNMAP,SECONDARY,QCFAIL,DUP]
-x, --ignore-overlaps
Disable read-pair overlap detection.
Output Options:
-o, --output FILE
Write pileup or VCF/BCF output to FILE, rather than
the default of standard output.
(The same short option is used for both --open-prob
and --output. If -o's argument contains any non-
digit characters other than a leading + or - sign,
it is interpreted as --output. Usually the file‐
name extension will take care of this, but to write
to an entirely numeric filename use -o ./123 or
--output 123.)
-g, --BCF Compute genotype likelihoods and output them in the
binary call format (BCF). As of v1.0, this is BCF2
which is incompatible with the BCF1 format produced
by previous (0.1.x) versions of samtools.
-v, --VCF Compute genotype likelihoods and output them in the
variant call format (VCF). Output is bgzip-com‐
pressed VCF unless -u option is set.
Output Options for mpileup format (without -g or -v):
-O, --output-BP
Output base positions on reads.
-s, --output-MQ
Output mapping quality.
Output Options for VCF/BCF format (with -g or -v):
-D Output per-sample read depth [DEPRECATED - use -t
DP instead]
-S Output per-sample Phred-scaled strand bias P-value
[DEPRECATED - use -t SP instead]
-t, --output-tags LIST
Comma-separated list of FORMAT and INFO tags to
output (case-insensitive): AD (Allelic depth, FOR‐
MAT), INFO/AD (Total allelic depth, INFO), ADF
(Allelic depths on the forward strand, FORMAT),
INFO/ADF (Total allelic depths on the forward
strand, INFO), ADR (Allelic depths on the reverse
strand, FORMAT), INFO/ADR (Total allelic depths on
the reverse strand, INFO), DP (Number of high-qual‐
ity bases, FORMAT), DV (Deprecated in favor of AD;
Number of high-quality non-reference bases, FOR‐
MAT), DPR (Deprecated in favor of AD; Number of
high-quality bases for each observed allele, FOR‐
MAT), INFO/DPR (Number of high-quality bases for
each observed allele, INFO), DP4 (Deprecated in
favor of ADF and ADR; Number of high-quality ref-
forward, ref-reverse, alt-forward and alt-reverse
bases, FORMAT), SP (Phred-scaled strand bias P-
value, FORMAT) [null]
-u, --uncompressed
Generate uncompressed VCF/BCF output, which is pre‐
ferred for piping.
-V Output per-sample number of non-reference reads
[DEPRECATED - use -t DV instead]
Options for SNP/INDEL Genotype Likelihood Computation (for -g
or -v):
-e, --ext-prob INT
Phred-scaled gap extension sequencing error proba‐
bility. Reducing INT leads to longer indels. [20]
-F, --gap-frac FLOAT
Minimum fraction of gapped reads [0.002]
-h, --tandem-qual INT
Coefficient for modeling homopolymer errors. Given
an l-long homopolymer run, the sequencing error of
an indel of size s is modeled as INT*s/l. [100]
-I, --skip-indels
Do not perform INDEL calling
-L, --max-idepth INT
Skip INDEL calling if the average per-sample depth
is above INT. [250]
-m, --min-ireads INT
Minimum number gapped reads for indel candidates
INT. [1]
-o, --open-prob INT
Phred-scaled gap open sequencing error probability.
Reducing INT leads to more indel calls. [40]
(The same short option is used for both --open-prob
and --output. When -o's argument contains only an
optional + or - sign followed by the digits 0 to 9,
it is interpreted as --open-prob.)
-p, --per-sample-mF
Apply -m and -F thresholds per sample to increase
sensitivity of calling. By default both options
are applied to reads pooled from all samples.
-P, --platforms STR
Comma-delimited list of platforms (determined by
@RG-PL) from which indel candidates are obtained.
It is recommended to collect indel candidates from
sequencing technologies that have low indel error
rate such as ILLUMINA. [all]
flags samtools flags INT|STR[,...]
Convert between textual and numeric flag representation.
FLAGS:
0x1 PAIRED paired-end (or multiple-segment) sequencing technology
0x2 PROPER_PAIR each segment properly aligned according to the aligner
0x4 UNMAP segment unmapped
0x8 MUNMAP next segment in the template unmapped
0x10 REVERSE SEQ is reverse complemented
0x20 MREVERSE SEQ of the next segment in the template is reverse complemented
0x40 READ1 the first segment in the template
0x80 READ2 the last segment in the template
0x100 SECONDARY secondary alignment
0x200 QCFAIL not passing quality controls
0x400 DUP PCR or optical duplicate
0x800 SUPPLEMENTARY supplementary 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.
OPTIONS:
-n By default, either '/1' or '/2' is added to the end
of read names where the corresponding BAM_READ1 or
BAM_READ2 flag is set. Using -n causes read names to
be left as they are.
-O Use quality values from OQ tags in preference to
standard quality string if available.
-s FILE Write singleton reads in FASTQ format to FILE instead
of outputting them.
-t Copy RG, BC and QT tags to the FASTQ header line, if
they exist.
-1 FILE Write reads with the BAM_READ1 flag set to FILE
instead of outputting them.
-2 FILE Write reads with the BAM_READ2 flag set to FILE
instead of outputting them.
-0 FILE Write reads with both or neither of the BAM_READ1 and
BAM_READ2 flags set to FILE instead of outputting
them.
-f INT Only output alignments with all bits set in INT
present in the FLAG field. INT can be specified in
hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/) or
in octal by beginning with `0' (i.e. /^0[0-7]+/) [0].
-F INT Do not output alignments with any bits set in INT
present in the FLAG field. INT can be specified in
hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/) or
in octal by beginning with `0' (i.e. /^0[0-7]+/) [0].
collate samtools collate [options] in.sam|in.bam|in.cram [out.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 oper‐
ation that requires all reads from the same template to be
grouped together.
Options:
-O Output to stdout rather than to files starting with
out.prefix
-u Write uncompressed BAM output
-l INT Compression level. [1]
-n INT Number of temporary files to use. [64]
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 stan‐
dard 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.
OPTIONS:
-P, --no-PG
Do not generate an @PG header line.
-i, --in-place
Perform the header edit in-place, if possible. This
only works on CRAM files and only if there is suffi‐
cient room to store the new header. The amount of
space available will differ for each CRAM file.
cat samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam>
[ ... ]
Concatenate BAMs. The sequence dictionary of each input BAM
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>
Remove potential PCR duplicates: if multiple read pairs have
identical external coordinates, only retain the pair with
highest mapping quality. In the paired-end mode, this com‐
mand ONLY works with FR orientation and requires ISIZE is
correctly set. It does not work for unpaired reads (e.g. two
ends mapped to different chromosomes or orphan reads).
OPTIONS:
-s Remove duplicates for single-end reads. By default,
the command works for paired-end reads only.
-S Treat paired-end reads and single-end reads.
addreplacerg
samtools addreplacerg [-r rg line | -R rg ID] [-m mode] [-l
level] [-o out.bam] <input.bam>
Adds or replaces read group tags in a file.
OPTIONS:
-r STRING
Allows you to specify a read group line to append to
the header and applies it to the reads specified by
the -m option. If repeated it automatically adds in
tabs between invocations.
-R STRING
Allows you to specify the read group ID of an exist‐
ing @RG line and applies it to the reads specified.
-m MODE If you choose orphan_only then existing RG tags are
not overwritten, if you choose overwrite_all, exist‐
ing RG tags are overwritten. The default is over‐
write_all.
-o STRING
Write the final output to STRING. The default is to
write to stdout.
By default, samtools tries to select a format based
on the output filename extension; if output is to
standard output or no format can be deduced, bam is
selected.
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 dif‐
ferent 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.
OPTIONS:
-A When used jointly with -r this option overwrites the
original base quality.
-e Convert a the read base to = if it is identical to
the aligned reference base. Indel caller does not
support the = bases at the moment.
-u Output uncompressed BAM
-b Output compressed BAM
-C INT Coefficient to cap mapping quality of poorly mapped
reads. See the pileup command for details. [0]
-r Compute the BQ tag (without -A) or cap base quality
by BAQ (with -A).
-E Extended BAQ calculation. This option trades speci‐
ficity for sensitivity, though the effect is minor.
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 con‐
tinuity 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.
OPTIONS:
-A Drop reads with ambiguous phase.
-b STR Prefix of BAM output. When this option is in use,
phase-0 reads will be saved in file STR.0.bam and
phase-1 reads in STR.1.bam. Phase unknown reads will
be randomly allocated to one of the two files.
Chimeric reads with switch errors will be saved in
STR.chimeric.bam. [null]
-F Do not attempt to fix chimeric reads.
-k INT Maximum length for local phasing. [13]
-q INT Minimum Phred-scaled LOD to call a heterozygote. [40]
-Q INT Minimum base quality to be used in het calling. [13]
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 consid‐
ered 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 dele‐
tions and padding cigar operations.
OPTIONS:
-S Ignored for compatibility with previous samtools ver‐
sions. Previously this option was required if input
was in SAM format, but now the correct format is auto‐
matically detected by examining the first few charac‐
ters of input.
-s Output in SAM format. The default is BAM.
-C Output in CRAM format. The default is BAM.
-u Do not compress the output. Applies to either BAM or
CRAM output format.
-1 Enable fastest compression level. Only works for BAM
or CRAM output.
-T FILE
Provides the padded reference file. Note that without
this the @SQ line lengths will be incorrect, so for
most use cases this option will be considered as
mandatory.
-o FILE
Specifies the output filename. By default output is
sent to stdout.
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 par‐
ticular 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-read‐
able format.
GLOBAL OPTIONS
Several long-options are shared between multiple samtools subcommands:
--input-fmt, --input-fmt-options, --output-fmt, --output-fmt-options,
and --reference. The input format is typically auto-detected so speci‐
fying 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-options 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 bool‐
ean option, the value is assumed to be 1. The valid options are as
follows.
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 decod‐
ing. It usually is not required for decoding except in the situa‐
tion of the MD5 not being obtainable via the REF_PATH or REF_CACHE
environment variables.
decode_md=0|1
CRAM decode only; defaults to 1 (on). CRAM does not typically
store MD and NM tags, preferring to generate them on the fly. This
option controls this behaviour.
ignore_md5=0|1
CRAM decode 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 decode 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.
0x1 SAM_QNAME
0x2 SAM_FLAG
0x4 SAM_RNAME
0x8 SAM_POS
0x10 SAM_MAPQ
0x20 SAM_CIGAR
0x40 SAM_RNEXT
0x80 SAM_PNEXT
0x100 SAM_TLEN
0x200 SAM_SEQ
0x400 SAM_QUAL
0x800 SAM_AUX
0x1000 SAM_RGAUX
multi_seq_per_slice=0|1
CRAM encode 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 encode only. Specifies the CRAM version number. Acceptable
values are "2.1" and "3.0".
seqs_per_slice=INT
CRAM encode only; defaults to 10000.
slices_per_container=INT
CRAM encode 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 encode only; defaults to 0 (off). If 1, this will store por‐
tions of the reference sequence in each slice, permitting decode
without having requiring an external copy of the reference
sequence.
no_ref=0|1
CRAM encode 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.
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 ref‐
erence 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 direc‐
tories 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 tempo‐
rary directory if no home directory is found) will be used simi‐
larly.
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 refer‐
ence it will be looked for in this directory before searching
REF_PATH. To avoid many files being stored in the same direc‐
tory, 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 distribu‐
tion. 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 -bS 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
o Attach the RG tag while merging sorted alignments:
perl -e 'print "@RG\tID:ga\tSM:hs\tLB:ga\tPL:Illumina\n@RG\tID:454\tSM:hs\tLB:454\tPL:454\n"' > rg.txt
samtools merge -rh rg.txt merged.bam ga.bam 454.bam
The value in a RG tag is determined by the file name the read is com‐
ing from. In this example, in the merged.bam, reads from ga.bam will
be attached RG:Z:ga, while reads from 454.bam will be attached
RG:Z:454.
o Call SNPs and short INDELs:
samtools mpileup -uf ref.fa aln.bam | bcftools call -mv > var.raw.vcf
bcftools filter -s LowQual -e '%QUAL<20 || DP>100' var.raw.vcf > var.flt.vcf
The bcftools filter command marks low quality sites and sites with
the read depth exceeding a limit, which should be adjusted to about
twice the average read depth (bigger read depths usually indicate
problematic regions which are often enriched for artefacts). One may
consider to add -C50 to mpileup if mapping quality is overestimated
for reads containing excessive mismatches. Applying this option usu‐
ally helps BWA-short but may not other mappers.
Individuals are identified from the SM tags in the @RG header lines.
Individuals can be pooled in one alignment file; one individual can
also be separated into multiple files. The -P option specifies that
indel candidates should be collected only from read groups with the
@RG-PL tag set to ILLUMINA. Collecting indel candidates from reads
sequenced by an indel-prone technology may affect the performance of
indel calling.
o Generate the consensus sequence for one diploid individual:
samtools mpileup -uf ref.fa aln.bam | bcftools call -c | vcfutils.pl vcf2fq > cns.fq
o Phase one individual:
samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out
The calmd command is used to reduce false heterozygotes around
INDELs.
o Dump BAQ applied alignment for other SNP callers:
samtools calmd -bAr aln.bam > aln.baq.bam
It adds and corrects the NM and MD tags at the same time. The calmd
command also comes with the -C option, the same as the one in pileup
and mpileup. Apply if it helps.
LIMITATIONS
o Unaligned words used in bam_import.c, bam_endian.h, bam.c and
bam_aux.c.
o Samtools paired-end rmdup does not work for unpaired reads (e.g.
orphan reads or ends mapped to different chromosomes). If this is a
concern, please use Picard's MarkDuplicates which correctly handles
these cases, although a little slower.
AUTHOR
Heng Li from the Sanger Institute wrote the original C version of sam‐
tools. Bob Handsaker from the Broad Institute implemented the BGZF
library. James Bonfield from the Sanger Institute developed the CRAM
implementation. John Marshall and Petr Danecek contribute to the
source code and various people from the 1000 Genomes Project have con‐
tributed to the SAM format specification.
SEE ALSObcftools(1), sam(5), tabix(1)
Samtools website: <http://www.htslib.org/>
File format specification of SAM/BAM,CRAM,VCF/BCF: <http://sam‐
tools.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>
samtools-1.3 15 December 2015 samtools(1)
