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 aln.bam aln.sorted
samtools index 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 pileup -f ref.fasta aln.sorted.bam
samtools tview aln.sorted.bam ref.fasta
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
import samtools import <in.ref_list> <in.sam> <out.bam>
Since 0.1.4, this command is an alias of:
samtools view -bt <in.ref_list> -o <out.bam> <in.sam>
sort samtools sort [-n] [-m maxMem] <in.bam> <out.prefix>
Sort alignments by leftmost coordinates. File
<out.prefix>.bam will be created. This command may also
create temporary files <out.prefix>.%d.bam when the whole
alignment cannot be fitted into memory (controlled by option
-m).
OPTIONS:
-n Sort by read names rather than by chromosomal
coordinates
-m INT Approximately the maximum required memory.
[500000000]
merge samtools merge [-h inh.sam] [-n] <out.bam> <in1.bam>
<in2.bam> [...]
Merge multiple sorted alignments. The header reference lists
of all the input BAM files, and the @SQ headers of inh.sam,
if any, must all refer to the same set of reference
sequences. The header reference list and (unless overridden
by -h) ‘@’ headers of in1.bam will be copied to out.bam, and
the headers of other files will be ignored.
OPTIONS:
-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
index samtools index <aln.bam>
Index sorted alignment for fast random access. Index file
<aln.bam>.bai will be created.
view samtools view [-bhuHS] [-t in.refList] [-o output] [-f
reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r
readGroup] <in.bam>|<in.sam> [region1 [...]]
Extract/print all or sub alignments in SAM or BAM format. If
no region is specified, all the alignments will be printed;
otherwise only alignments overlapping the specified regions
will be output. An alignment may be given multiple times if
it is overlapping several regions. A region can be presented,
for example, in the following format: ‘chr2’ (the whole
chr2), ‘chr2:1000000’ (region starting from 1,000,000bp) or
‘chr2:1,000,000-2,000,000’ (region between 1,000,000 and
2,000,000bp including the end points). The coordinate is
1-based.
OPTIONS:
-b Output in the BAM format.
-u Output uncompressed BAM. This option saves time spent
on compression/decomprssion 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.
-S Input is in SAM. If @SQ header lines are absent, the
‘-t’ option is required.
-t FILE This file is TAB-delimited. Each line must contain
the reference name and the length of the reference,
one line for each distinct reference; additional
fields are ignored. This file also defines the order
of the reference sequences in sorting. If you run
‘samtools faidx <ref.fa>’, the resultant index file
<ref.fa>.fai can be used as this <in.ref_list> file.
-o FILE Output file [stdout]
-f INT Only output alignments with all bits in INT present
in the FLAG field. INT can be in hex in the format of
/^0x[0-9A-F]+/ [0]
-F INT Skip alignments with bits present in INT [0]
-q INT Skip alignments with MAPQ smaller than INT [0]
-l STR Only output reads in library STR [null]
-r STR Only output reads in read group STR [null]
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 speficified, the
subsequences will be retrieved and printed to stdout in the
FASTA format. The input file can be compressed in the RAZF
format.
pileup samtools pileup [-f in.ref.fasta] [-t in.ref_list] [-l
in.site_list] [-iscgS2] [-T theta] [-N nHap] [-r
pairDiffRate] <in.bam>|<in.sam>
Print the alignment in the pileup format. In the pileup
format, each line represents a genomic position, consisting
of chromosome name, coordinate, reference base, read bases,
read 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, ‘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 segment which is a contiguous
subsequence on the read separated by ‘N/S/H’ CIGAR
operations. The ASCII of the character following ‘^’ minus 33
gives the mapping quality. A symbol ‘$’ marks the end of a
read segment.
If option -c is applied, the consensus base, Phred-scaled
consensus quality, SNP quality (i.e. the Phred-scaled
probability of the consensus being identical to the
reference) and root mean square (RMS) mapping quality of the
reads covering the site will be inserted between the
‘reference base’ and the ‘read bases’ columns. An indel
occupies an additional line. Each indel line consists of
chromosome name, coordinate, a star, the genotype, consensus
quality, SNP quality, RMS mapping quality, # covering reads,
the first alllele, the second allele, # reads supporting the
first allele, # reads supporting the second allele and #
reads containing indels different from the top two alleles.
OPTIONS:
-s Print the mapping quality as the last column. This
option makes the output easier to parse, although
this format is not space efficient.
-S The input file is in SAM.
-i Only output pileup lines containing indels.
-f FILE The reference sequence in the FASTA format. Index
file FILE.fai will be created if absent.
-M INT Cap mapping quality at INT [60]
-t FILE List of reference names ane sequence lengths, in
the format described for the import command. If
this option is present, samtools assumes the input
<in.alignment> is in SAM format; otherwise it
assumes in BAM format.
-l FILE List of sites at which pileup is output. This file
is space delimited. The first two columns are
required to be chromosome and 1-based coordinate.
Additional columns are ignored. It is recommended
to use option -s together with -l as in the default
format we may not know the mapping quality.
-c Call the consensus sequence using MAQ consensus
model. Options -T, -N, -I and -r are only effective
when -c or -g is in use.
-g Generate genotype likelihood in the binary GLFv3
format. This option suppresses -c, -i and -s.
-T FLOAT The theta parameter (error dependency coefficient)
in the maq consensus calling model [0.85]
-N INT Number of haplotypes in the sample (>=2) [2]
-r FLOAT Expected fraction of differences between a pair of
haplotypes [0.001]
-I INT Phred probability of an indel in sequencing/prep.
[40]
tview samtools tview <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’.
fixmate samtools fixmate <in.nameSrt.bam> <out.bam>
Fill in mate coordinates, ISIZE and mate related flags from a
name-sorted alignment.
rmdup samtools rmdup <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. This command ONLY works with FR
orientation and requires ISIZE is correctly set.
rmdupse samtools rmdupse <input.srt.bam> <out.bam>
Remove potential duplicates for single-ended reads. This
command will treat all reads as single-ended even if they are
paired in fact.
fillmd samtools fillmd [-e] <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.
OPTIONS:
-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.
SAM FORMAT
SAM is TAB-delimited. Apart from the header lines, which are started
with the ‘@’ symbol, each alignment line consists of:
+----+-------+----------------------------------------------------------+
|Col | Field | Description |
+----+-------+----------------------------------------------------------+
| 1 | QNAME | Query (pair) NAME |
| 2 | FLAG | bitwise FLAG |
| 3 | RNAME | Reference sequence NAME |
| 4 | POS | 1-based leftmost POSition/coordinate of clipped sequence |
| 5 | MAPQ | MAPping Quality (Phred-scaled) |
| 6 | CIAGR | extended CIGAR string |
| 7 | MRNM | Mate Reference sequence NaMe (‘=’ if same as RNAME) |
| 8 | MPOS | 1-based Mate POSistion |
| 9 | ISIZE | Inferred insert SIZE |
|10 | SEQ | query SEQuence on the same strand as the reference |
|11 | QUAL | query QUALity (ASCII-33 gives the Phred base quality) |
|12 | OPT | variable OPTional fields in the format TAG:VTYPE:VALUE |
+----+-------+----------------------------------------------------------+
Each bit in the FLAG field is defined as:
+-------+--------------------------------------------------+
| Flag | Description |
+-------+--------------------------------------------------+
|0x0001 | the read is paired in sequencing |
|0x0002 | the read is mapped in a proper pair |
|0x0004 | the query sequence itself is unmapped |
|0x0008 | the mate is unmapped |
|0x0010 | strand of the query (1 for reverse) |
|0x0020 | strand of the mate |
|0x0040 | the read is the first read in a pair |
|0x0080 | the read is the second read in a pair |
|0x0100 | the alignment is not primary |
|0x0200 | the read fails platform/vendor quality checks |
|0x0400 | the read is either a PCR or an optical duplicate |
+-------+--------------------------------------------------+
LIMITATIONS
o Unaligned words used in bam_import.c, bam_endian.h, bam.c and
bam_aux.c.
o CIGAR operation P is not properly handled at the moment.
o In merging, the input files are required to have the same number of
reference sequences. The requirement can be relaxed. In addition,
merging does not reconstruct the header dictionaries automatically.
Endusers have to provide the correct header. Picard is better at
merging.
o Samtools’ rmdup does not work for single-end data and does not remove
duplicates across chromosomes. Picard is better.
AUTHOR
Heng Li from the Sanger Institute wrote the C version of samtools. Bob
Handsaker from the Broad Institute implemented the BGZF library and Jue
Ruan from Beijing Genomics Institute wrote the RAZF library. Various
people in the 1000Genomes Project contributed to the SAM format
specification.
SEE ALSO
Samtools website: <http://samtools.sourceforge.net>