samtools mpileup [-EB] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]]
Generate text pileup output for one or multiple BAM files. Each input file produces a separate group of pileup columns in the output.
Samtools mpileup can still produce VCF and BCF output (with -g or -u), 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/>.)
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.
Several columns contain numeric quality values encoded as individual ASCII characters. Each character can range from “!” to “~” and is decoded by taking its ASCII value and subtracting 33; e.g., “A” encodes the numeric value 32.
The first three columns give the position and reference:
The remaining columns show the pileup data, and are repeated for each input BAM file specified:
For each read covering the position, this column contains:
|. dot||, comma||Base matches the reference base|
|ACGTN||acgtn||Base is a mismatch to the reference base|
|>||<||Reference skip (due to CIGAR “N”)|
|*||*/#||Deletion of the reference base (CIGAR “D”)|
Deleted bases are shown as “*” on both strands unless --reverse-del is used, in which case they are shown as “#” on the reverse strand.
Assume the quality is in the Illumina 1.3+ encoding.
Do not skip anomalous read pairs in variant calling. Anomalous read pairs are those marked in the FLAG field as paired in sequencing but without the properly-paired flag set.
List of input BAM files, one file per line [null]
Disable base alignment quality (BAQ) computation. See BAQ below.
Coefficient for downgrading mapping quality for reads containing excessive mismatches. Given a read with a phred-scaled probability q of being generated from the mapped position, the new mapping quality is about sqrt((INT-q)/INT)*INT. A zero value disables this functionality; if enabled, the recommended value for BWA is 50. 
At a position, read maximally INT reads per input file. Setting this limit reduces the amount of memory and time needed to process regions with very high coverage. Passing zero for this option sets it to the highest possible value, effectively removing the depth limit. 
Note that up to release 1.8, samtools would enforce a minimum value for this option. This no longer happens and the limit is set exactly as specified.
Recalculate BAQ on the fly, ignore existing BQ tags. See BAQ below.
The faidx-indexed reference file in the FASTA format. The file can be optionally compressed by bgzip. [null]
Supplying a reference file will enable base alignment quality calculation for all reads aligned to a reference in the file. See BAQ below.
Exclude reads from read groups listed in FILE (one @RG-ID per line)
BED or position list file containing a list of regions or sites where
pileup or BCF should be generated. Position list files contain two
columns (chromosome and position) and start counting from 1. BED
files contain at least 3 columns (chromosome, start and end position)
and are 0-based half-open.
While it is possible to mix both position-list and BED coordinates in the same file, this is strongly ill advised due to the differing coordinate systems. [null]
Minimum mapping quality for an alignment to be used 
Minimum base quality for a base to be considered 
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]
Ignore RG tags. Treat all reads in one BAM as one sample.
Required flags: include reads with any of the mask bits set [null]
Filter flags: skip reads with any of the mask bits set [UNMAP,SECONDARY,QCFAIL,DUP]
Disable read-pair overlap detection.
Include customized index file as a part of arguments. See EXAMPLES section for sample of usage.
Write pileup output to FILE, rather than the default of standard output.
(The same short option is used for both the deprecated --open-prob option and --output. If -o's argument contains any non-digit characters other than a leading + or - sign, it is interpreted as --output. Usually the filename extension will take care of this, but to write to an entirely numeric filename use -o ./123 or --output 123.)
Output base positions on reads.
Output mapping qualities encoded as ASCII characters.
Output an extra column containing comma-separated read names. Equivalent to --output-extra QNAME.
Output extra columns containing comma-separated values of read fields or read tags. The names of the selected fields have to be provided as they are described in the SAM Specification (pag. 6) and will be output by the mpileup command in the same order as in the document (i.e. QNAME, FLAG, RNAME,...) The names are case sensitive. Currently, only the following fields are supported:
QNAME, FLAG, RNAME, POS, MAPQ, RNEXT, PNEXT
Anything that is not on this list is treated as a potential tag, although only two character tags are accepted. In the mpileup output, tag columns are displayed in the order they were provided by the user in the command line. Field and tag names have to be provided in a comma-separated string to the mpileup command. E.g.
samtools mpileup --output-extra FLAG,QNAME,RG,NM in.bam
will display four extra columns in the mpileup output, the first being a list of comma-separated read names, followed by a list of flag values, a list of RG tag values and a list of NM tag values. Field values are always displayed before tag values.
Specify a different separator character for tag value lists, when those values might contain one or more commas (,), which is the default list separator. This option only affects columns for two-letter tags like NM; standard fields like FLAG or QNAME will always be separated by commas.
Specify a different 'no value' character for tag list entries corresponding to reads that don't have a tag requested with the --output-extra option. The default is *.
This option only applies to rows that have at least one read in the pileup, and only to columns for two-letter tags. Columns for empty rows will always be printed as *.
Mark the deletions on the reverse strand with the character #, instead of the usual *.
Output all positions, including those with zero depth.
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.
BAQ (Base Alignment Quality)
BAQ is the Phred-scaled probability of a read base being misaligned. It greatly helps to reduce false SNPs caused by misalignments. BAQ is calculated using the probabilistic realignment method described in the paper “Improving SNP discovery by base alignment quality”, Heng Li, Bioinformatics, Volume 27, Issue 8 <https://doi.org/10.1093/bioinformatics/btr076>
BAQ is turned on when a reference file is supplied using the -f option. To disable it, use the -B option.
It is possible to store precalculated BAQ values in a SAM BQ:Z tag. Samtools mpileup will use the precalculated values if it finds them. The -E option can be used to make it ignore the contents of the BQ:Z tag and force it to recalculate the BAQ scores by making a new alignment.
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.vcfThe 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.
samtools mpileup -uf ref.fa aln.bam | bcftools call -c | vcfutils.pl vcf2fq > cns.fq
samtools mpileup [options] -X /data_folder/in1.bam [/data_folder/in2.bam [...]] /index_folder/index1.bai [/index_folder/index2.bai [...]]
samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.outThe calmd command is used to reduce false heterozygotes around INDELs.
Written by Heng Li from the Sanger Institute.
samtools (1), samtools-depth (1), samtools-sort (1), bcftools (1)
Samtools website: <http://www.htslib.org/>
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