NAME

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

SYNOPSIS

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

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

samtools index aln.sorted.bam

samtools idxstats aln.sorted.bam

samtools flagstat aln.sorted.bam

samtools stats aln.sorted.bam

samtools bedcov aln.sorted.bam

samtools depth aln.sorted.bam

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

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

samtools faidx ref.fasta

samtools fqidx ref.fastq

samtools tview aln.sorted.bam ref.fasta

samtools split merged.bam

samtools quickcheck in1.bam in2.cram

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

samtools fixmate in.namesorted.sam out.bam

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

samtools flags PAIRED,UNMAP,MUNMAP

samtools fastq input.bam > output.fastq

samtools fasta input.bam > output.fasta

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

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

samtools depad input.bam

samtools markdup in.algnsorted.bam out.bam

DESCRIPTION

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

OPTIONS:

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:

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:

idxstats

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

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

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

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

flagstat

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

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

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

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:

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:

bedcov

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

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

Options:

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:

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:

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.

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

Options

fqidx

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

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

The input file can be compressed in the BGZF format.

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.

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

Options

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:

split

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

Splits a file by read group.

Options:

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:

dict

samtools dict <ref.fasta|ref.fasta.gz>

Create a sequence dictionary file from a fasta file.

OPTIONS:

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:

mpileup

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

Generate 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.

Samtools mpileup can still produce VCF and BCF output, but this feature is deprecated and will be removed in a future release. Please use bcftools mpileup for this instead. (Documentation on the deprecated options has been removed from this manual page, but older versions are available online at <http://www.htslib.org/doc/>.)

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:

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 files will be automatically compressed if the file names have a .gz or .bgzf extension.

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

For each different QNAME, the input records are categorised according to the state of the READ1 and READ2 flag bits. The three categories used are:

1 : Only READ1 is set.

2 : Only READ2 is set.

0 : Either both READ1 and READ2 are set; or neither is set.

The exact meaning of these categories depends on the sequencing technology used. It is expected that ordinary single and paired-end sequencing reads will be in categories 1 and 2 (in the case of paired-end reads, one read of the pair will be in category 1, the other in category 2). Category 0 is essentially a “catch-all” for reads that do not fit into a simple paired-end sequencing model.

For each category only one sequence will be written for a given QNAME. If more than one record is available for a given QNAME and category, the first in input file order that has quality values will be used. If none of the candidate records has quality values, then the first in input file order will be used instead.

Sequences will be written to standard output unless one of the -1,-2, or -0 options is used, in which case sequences for that category will be written to the specified file.

If a singleton file is specified using the -s option then only paired sequences will be output for categories 1 and 2; paired meaning that for a given QNAME there are sequences for both category 1 and 2. If there is a sequence for only one of categories 1 or 2 then it will be diverted into the specified singletons file. This can be used to prepare fastq files for programs that cannot handle a mixture of paired and singleton reads.

The -s option only affects category 1 and 2 records. The output for category 0 will be the same irrespective of the use of this option.

OPTIONS:

samtools fastq -1 paired1.fq -2 paired2.fq -0 /dev/null -s /dev/null -n -F 0x900 in.bam

samtools fastq -0 /dev/null -F 0x900 in.bam > all_reads.fq

samtools fastq -0 /dev/null -s single.fq -N -F 0x900 in.bam > paired.fq

collate

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

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

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

If present, <prefix> is used to name the temporary files that collate uses when sorting the data. If neither the '-O' nor '-o' options are used, <prefix> must be present and collate will use it to make an output file name by appending a suffix depending on the format written (.bam by default).

If either the -O or -o option is used, <prefix> is optional. If <prefix> is absent, collate will write the temporary files to a system-dependent location (/tmp on UNIX).

Using -f for fast mode will output only primary alignments that have either the READ1 or READ2 flags set (but not both). Any other alignment records will be filtered out. The collation will only work correctly if there are no more than two reads for any given QNAME after filtering.

Fast mode keeps a buffer of alignments in memory so that it can write out most pairs as soon as they are found instead of storing them in temporary files. This allows collate to avoid some work and so finish more quickly compared to the standard mode. The number of alignments held can be changed using -r, storing more alignments uses more memory but increases the number of pairs that can be written early.

While collate normally randomises the ordering of read pairs, fast mode does not. Position-dependent biases that would normally be broken up can remain in the fast collate output. It is therefore not a good idea to use fast mode when preparing data for programs that expect randomly ordered paired reads. For example using fast collate instead of the standard mode may lead to significantly different results from aligners that estimate library insert sizes on batches of reads.

Options:

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:

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:

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:

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:

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:

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:

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:

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.

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-option, --output-fmt, --output-fmt-option, and --reference. The input format is typically auto-detected so specifying the format is usually unnecessary and the option is included for completeness. Note that not all subcommands have all options. Consult the subcommand help for more details.

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

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

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

Convert a BAM file to a CRAM with NM and MD tags stored verbatim rather than calculating on the fly during CRAM decode, so that mixed data sets with MD/NM only on some records, or NM calculated using different definitions of mismatch, can be decoded without change. The second command demonstrates how to decode such a file. The request to not decode MD here is turning off auto-generation of both MD and NM; it will still emit the MD/NM tags on records that had these stored verbatim.

samtools view -C --output-fmt-option store_md=1 --output-fmt-option store_nm=1 -o aln.cram aln.bam
samtools view --input-fmt-option decode_md=0 -o aln.new.bam aln.cram
    

o

An alternative way of achieving the above is listing multiple options after the --output-fmt or -O option. The commands below are equivalent to the two above.

samtools view -O cram,store_md=1,store_nm=1 -o aln.cram aln.bam
samtools view --input-fmt cram,decode_md=0 -o aln.new.bam aln.cram
    

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.

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Generate the consensus sequence for one diploid individual:

samtools mpileup -uf ref.fa aln.bam | bcftools call -c | vcfutils.pl vcf2fq > cns.fq
    

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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.

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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

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

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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>