Provided by: samtools_1.7-1_amd64 
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 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 output.bam input.bam
samtools collate aln.sorted.bam aln.name_collated.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 one 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 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 (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 contain @SQ headers, and the -T
option is required whenever writing CRAM output.
The -L, -M, -r, -R, -s, -q, -l, -m, -f, -F, and -G options filter the alignments
that will be included in the output to only those alignments that match certain
criteria.
The -x and -B 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 alignments 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 position 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 preferred 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 various filter options
to FILE. When this option is used, all alignments (or all alignments
intersecting 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 compressed 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].
-M Use the multi-region iterator on the union of the BED file and
command-line region arguments. This avoids re-reading the same
regions of files so can sometimes be much faster. Note this also
removes duplicate sequences. Without this a sequence that overlaps
multiple regions specified on the command line will be reported
multiple times.
-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].
-G INT Do not output alignments with all bits set in INT present in the FLAG
field. This is the opposite of -f such that -f12 -G12 is the same as
no filtering at all. 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 Output only a proportion of the input alignments. This subsampling
acts in the same way on all of the alignment records in the same
template or read pair, so it never keeps a read but not its mate.
The integer and fractional parts of the -s INT.FRAC option are used
separately: the part after the decimal point sets the fraction of
templates/pairs to be kept, while the integer part is used as a seed
that influences which subset of reads is kept.
When subsampling data that has previously been subsampled, be sure to
use a different seed value from those used previously; otherwise more
reads will be retained than expected.
-@ INT Number of BAM compression threads to use in addition 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 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).
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 compression 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]
To prevent sort from creating a huge number of temporary files, it
enforces a minimum value of 1M for this setting.
-n Sort by read names (i.e., the QNAME field) rather than by chromosomal
coordinates.
-t TAG Sort first by the value in the alignment tag TAG, then by position or
name (if also using -n). -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, or if the specified PREFIX
is an existing directory, to PREFIX/samtools.mmm.mmm.tmp.nnnn.bam,
where mmm is unique to this invocation of the sort command.
By default, any temporary files are written alongside the output
file, as out.bam.tmp.nnnn.bam, or if output is to standard output, in
the current directory as samtools.mmm.mmm.tmp.nnnn.bam.
-@ INT Set number of sorting and compression threads. By default, operation
is single-threaded.
Ordering Rules
The following rules are used for ordering records.
If option -t is in use, records are first sorted by the value of the given
alignment tag, and then by position or name (if using -n). For example, “-t RG”
will make read group the primary sort key. The rules for ordering by tag are:
• Records that do not have the tag are sorted before ones that do.
• If the types of the tags are different, they will be sorted so that single
character tags (type A) come before array tags (type B), then string tags
(types H and Z), then numeric tags (types f and i).
• Numeric tags (types f and i) are compared by value. Note that comparisons
of floating-point values are subject to issues of rounding and precision.
• String tags (types H and Z) are compared based on the binary contents of the
tag using the C strcmp(3) function.
• Character tags (type A) are compared by binary character value.
• No attempt is made to compare tags of other types — notably type B array
values will not be compared.
When the -n option is present, records are sorted by name. Names are compared
so as to give a “natural” ordering — i.e. sections consisting of digits are
compared numerically while all other sections are compared based on their binary
representation. This means “a1” will come before “b1” and “a9” will come before
“a10”. Records with the same name will be ordered according to the values of
the READ1 and READ2 flags (see flags).
When the -n option is not present, reads are sorted by reference (according to
the order of the @SQ header records), then by position in the reference, and
then by the REVERSE flag.
Note
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 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.
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 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.
The first row of output gives the total number of reads that are QC pass and
fail (according to flag bit 0x200). For example:
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 output 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 mismatches-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 insertions.
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. Counts for each
alignment file supplied are reported in separate columns.
Options:
-Q INT Only count reads with mapping quality greater than INT
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.
Note that when used in conjunction with a BED file the -a option may
sometimes operate as if -aa was specified if the reference sequence has
coverage outside of the region specified in the BED file.
-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 headers. 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 differentiate them from
similar header records from other files and the read records will be updated to
reflect this.
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.
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 otherwise be copied from in1.bam. (FILE is
actually in SAM format, though any alignment records it may contain are
ignored.)
-n The input alignments are sorted by read names rather than by chromosomal
coordinates
-t TAG The input alignments have been sorted by the value of TAG, then by
either position or name (if -n is given).
-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
suffixes 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
differentiate similar IDs.
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.
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 subsequences 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 alignment 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 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).
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.
-q Quiet mode: disables warning messages on stderr about files that fail.
If both -q and -v options are used then the appropriate level of -v
takes precedence.
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 [-rpcm] [-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.
-m Add ms (mate score) tags. These are used by markdup to select the
best reads to keep.
-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 qualities. 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]+[ACGTNacgtn]+' indicates there is an
insertion between this reference position and the next reference position. The
length of the insertion is given by the integer in the pattern, followed by the
inserted sequence. Similarly, a pattern `-[0-9]+[ACGTNacgtn]+' represents a
deletion from the reference. The deleted bases will be presented as `*' in the
following lines. Also at the read base column, a symbol `^' marks the start of a
read. The ASCII of the character following `^' minus 33 gives the mapping
quality. A symbol `$' marks the end of a read segment.
Note that there are two orthogonal ways to specify locations in the input file;
via -r region and -l file. The former uses (and requires) an index to do random
access while the latter streams through the file contents filtering out the
specified regions, requiring no index. The two may be used in conjunction. For
example a BED file containing locations of genes in chromosome 20 could be
specified using -r 20 -l chr20.bed, meaning that the index is used to find
chromosome 20 and then it is filtered for the regions listed in the bed file.
Input Options:
-6, --illumina1.3+
Assume the quality is in the Illumina 1.3+ encoding.
-A, --count-orphans
Do not skip anomalous read pairs in variant calling.
-b, --bam-list FILE
List of input BAM files, one file per line [null]
-B, --no-BAQ
Disable probabilistic realignment for the computation of base
alignment quality (BAQ). BAQ is the Phred-scaled probability of a read
base being misaligned. 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 generated 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 file. Note that
samtools has a minimum value of 8000/n where n is the number of input
files given to mpileup. This means the default is highly likely to be
increased. Once above the cross-sample minimum of 8000 the -d
parameter will have an effect. [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 format. 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 generated. Position list files contain two
columns (chromosome and position) and start counting from 1. BED
files contain at least 3 columns (chromosome, start and end position)
and are 0-based half-open.
While it is possible to mix both position-list and BED coordinates in
the same file, this is strongly ill advised due to the differing
coordinate systems. [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 filename
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-compressed 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-QNAME
Output an extra column containing comma-separated read names.
-a Output all positions, including those with zero depth.
-a -a, -aa
Output absolutely all positions, including unused reference sequences.
Note that when used in conjunction with a BED file the -a option may
sometimes operate as if -aa was specified if the reference sequence
has coverage outside of the region specified in the BED file.
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, FORMAT), 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-
quality bases, FORMAT), DV (Deprecated in favor of AD; Number of high-
quality non-reference bases, FORMAT), DPR (Deprecated in favor of AD;
Number of high-quality bases for each observed allele, FORMAT),
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 preferred 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 probability. 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-input-file 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. The FASTQ files will be automatically compressed if the
filenames have a .gz or .bgzf extention.
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.
-N Always add either '/1' or '/2' to the end of read names even when put
into different files.
-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.
-T TAGLIST
Specify a comma-separated list of tags to copy 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].
-G INT Only EXCLUDE reads with all of the 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].
-i add Illumina Casava 1.8 format entry to header (eg 1:N:0:ATCACG)
-c [0..9]
set compression level when writing gz or bgzf fastq files.
--i1 FILE
write first index reads to FILE
--i2 FILE
write second index reads to FILE
--barcode-tag TAG
aux tag to find index reads in [default: BC]
--quality-tag TAG
aux tag to find index quality in [default: QT]
--index-format STR
string to describe how to parse the barcode and quality tags. For
example:
i14i8 the first 14 characters are index 1, the next 8 characters are
index 2
n8i14 ignore the first 8 characters, and use the next 14 characters
for index 1
If the tag contains a separator, then the numeric part can be
replaced with '*' to mean 'read until the separator or end of
tag', for example:
n*i* ignore the left part of the tag until the separator, then use
the second part
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 operation 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 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.
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 sufficient room to store the new header. The
amount of space available will differ for each CRAM file.
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.
OPTIONS:
-b FOFN Read the list of input BAM or CRAM files from FOFN. These are
concatenated prior to any files specified on the command line. Multiple
-b FOFN options may be specified to concatenate multiple lists of
BAM/CRAM files.
-h FILE Uses the SAM header from FILE. By default the header is taken from the
first file to be concatenated.
-o FILE Write the concatenated output to FILE. By default this is sent to
stdout.
rmdup samtools rmdup [-sS] <input.srt.bam> <out.bam>
This command is obsolete. Use markdup instead.
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 command 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 existing @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, existing RG tags are overwritten. The default
is overwrite_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 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.
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 specificity 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 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.
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 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.
OPTIONS:
-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.
-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.
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 fixmate with the -m option. This program relies on the MC and ms tags
that fixmate provides.
-l INT Expected maximum read length of INT bases. [300]
-r Remove duplicate reads.
-s Print some basic stats.
-T PREFIX Write temporary files to PREFIX.samtools.nnnn.mmmm.tmp
-S Mark supplementary reads of duplicates as duplicates.
EXAMPLE
# The first sort can be omitted if the file is already name ordered
samtools sort -n -o namesort.bam example.bam
# Add ms and MC tags for markdup to use later
samtools fixmate -m namesort.bam fixmate.bam
# Markdup needs position order
samtools sort -o positionsort.bam fixmate.bam
# Finally mark duplicates
samtools markdup positionsort.bam markdup.bam
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 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 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-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 boolean 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 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. This option controls this behaviour.
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.
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
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 -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 coming 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 usually 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 samtools. 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 contributed to the
SAM format specification.
SEE ALSO
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>