SAMTOOLS(1) - Linux man page online | User commands

Utilities for the Sequence Alignment/Map (SAM) format.

26 January 2018
samtools(1) Bioinformatics tools samtools(1)


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


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


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.


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 coordi‐ nates 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 compres‐ sion/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 ide‐ ally 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 com‐ mand-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 tem‐ plate 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 tem‐ plates/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 cor‐ rect format is automatically detected by examining the first few char‐ acters 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 com‐ pression 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 file‐ name 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 com‐ pared 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 qual‐ ity 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 require‐ ment) [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 cal‐ culation). [] -S, --split TAG In addition to the complete statistics, also output categorised statis‐ tics 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>.bam‐ stat, 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 align‐ ment 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 speci‐ fied 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 unde‐ fined. 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 usu‐ ally 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 suf‐ fixes 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 align‐ ment. 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 min‐ BaseQ] [-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 posi‐ tion, consisting of chromosome name, 1-based coordinate, reference base, the number of reads covering the site, read bases, base qualities and alignment map‐ ping 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 inser‐ tion 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 qual‐ ity. 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 spec‐ ified using -r 20 -l chr20.bed, meaning that the index is used to find chromo‐ some 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 align‐ ment 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 functional‐ ity; 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 sam‐ tools 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 parame‐ ter 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 coor‐ dinate 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,SEC‐ ONDARY,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 exten‐ sion will take care of this, but to write to an entirely numeric file‐ name 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 for‐ mat (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-insensi‐ tive): 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, FOR‐ MAT), DV (Deprecated in favor of AD; Number of high-quality non-refer‐ ence 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 homopoly‐ mer 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 call‐ ing. 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 can‐ didates 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 compleme ↲ nted 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 com‐ mand 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 exam‐ ple: 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 (std‐ out), 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 concate‐ nated 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 std‐ out. rmdup samtools rmdup [-sS] <> <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 cor‐ rectly set. It does not work for unpaired reads (e.g. two ends mapped to differ‐ ent 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 auto‐ matically 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 file‐ name 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 qual‐ ity. -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 sensitiv‐ ity, 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 pad‐ ding 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 con‐ sidered 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.


Several long-options are shared between multiple samtools subcommands: --input-fmt, --input-fmt-options, --output-fmt, --output-fmt-options, and --reference. The input for‐ mat 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 refer‐ ence 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 perfor‐ mance 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 refer‐ ence 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 compres‐ sion. 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


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.


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 sepa‐ rator. For URLs, the text %s will be replaced by the MD5sum being read. If no REF_PATH has been specified it will default to 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 subdi‐ rectories 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/ is provided in the Samtools distribution. This takes a fasta file or a directory of fasta files and generates the MD5sum named files.


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 (big‐ ger read depths usually indicate problematic regions which are often enriched for arte‐ facts). 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 | 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.


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 MarkDupli‐ cates which correctly handles these cases, although a little slower.


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.


bcftools(1), sam(5), tabix(1) Samtools website: <> File format specification of SAM/BAM,CRAM,VCF/BCF: <> Samtools latest source: <> HTSlib latest source: <> Bcftools website: <>
samtools-1.7 26 January 2018 samtools(1)
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