Ubuntu Manpage: samtools mpileup - produces "pileup" textual format from an alignment

NAME

       samtools mpileup - produces "pileup" textual format from an alignment

SYNOPSIS

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

DESCRIPTION

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.

Pileup Format
Pileup format consists of TAB-separated lines, with each line representing the pileup of
reads at a single genomic position.

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:

○ Chromosome name.

○ 1-based position on the chromosome.

○ Reference base at this position (this will be “N” on all lines if -f/--fasta-ref has not
been used).

The remaining columns show the pileup data, and are repeated for each input BAM file
specified:

○ Number of reads covering this position.

○ Read bases. This encodes information on matches, mismatches, indels, strand, mapping
quality, and starts and ends of reads.

For each read covering the position, this column contains:

• If this is the first position covered by the read, a “^” character followed by the
alignment's mapping quality encoded as an ASCII character.

• A single character indicating the read base and the strand to which the read has been
mapped:

Forward Reverse Meaning
───────────────────────────────────────────────────────────────

. 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.

• If there is an insertion after this read base, text matching
“\+[0-9]+[ACGTNacgtn*#]+”: a “+” character followed by an integer giving the length of
the insertion and then the inserted sequence. Pads are shown as “*” unless --reverse-
del is used, in which case pads on the reverse strand will be shown as “#”.

• If there is a deletion after this read base, text matching “-[0-9]+[ACGTNacgtn]+”: a
“-” character followed by the deleted reference bases represented similarly.
(Subsequent pileup lines will contain “*” for this read indicating the deleted bases.)

• If this is the last position covered by the read, a “$” character.

○ Base qualities, encoded as ASCII characters.

○ Alignment mapping qualities, encoded as ASCII characters. (Column only present when
-s/--output-MQ is used.)

○ Comma-separated 1-based positions within the alignments, e.g., 5 indicates that it is
the fifth base of the corresponding read that is mapped to this genomic position.
(Column only present when -O/--output-BP is used.)

○ Comma-separated read names. (Column only present when --output-QNAME is used.)

○ Additional columns containing other specified read fields or tags, as selected via the
--output-extra, --output-sep, and --output-empty options.

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. Anomolous read pairs are
those marked in the FLAG field as paired in sequencing but without the properly-
paired flag set.

-b, --bam-list FILE
List of input BAM files, one file per line [null]

-B, --no-BAQ
Disable base alignment quality (BAQ) computation. See BAQ below.

-C, --adjust-MQ INT
Coefficient for downgrading mapping quality for reads containing excessive
mismatches. Given a read with a phred-scaled probability q of being generated
from the mapped position, the new mapping quality is about sqrt((INT-
q)/INT)*INT. A zero value disables this functionality; if enabled, the
recommended value for BWA is 50. [0]

-d, --max-depth INT
At a position, read maximally INT reads per input file. 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. [8000]

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.

-E, --redo-BAQ
Recalculate BAQ on the fly, ignore existing BQ tags. See BAQ below.

-f, --fasta-ref FILE
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.

-G, --exclude-RG FILE
Exclude reads from readgroups listed in FILE (one @RG-ID per line)

-l, --positions FILE
BED or position list file containing a list of regions or sites where pileup or
BCF should be generated. Position list files contain two columns (chromosome and
position) and start counting from 1. BED files contain at least 3 columns
(chromosome, start and end position) and are 0-based half-open.
While it is possible to mix both position-list and BED coordinates in the same
file, this is strongly ill advised due to the differing coordinate systems.
[null]

-q, -min-MQ INT
Minimum mapping quality for an alignment to be used [0]

-Q, --min-BQ INT
Minimum base quality for a base to be considered [13]

-r, --region STR
Only generate pileup in region. Requires the BAM files to be indexed. If used
in conjunction with -l then considers the intersection of the two requests. STR
[all sites]

-R, --ignore-RG
Ignore RG tags. Treat all reads in one BAM as one sample.

--rf, --incl-flags STR|INT
Required flags: skip reads with mask bits unset [null]

--ff, --excl-flags STR|INT
Filter flags: skip reads with mask bits set [UNMAP,SECONDARY,QCFAIL,DUP]

-x, --ignore-overlaps
Disable read-pair overlap detection.

-X Include customized index file as a part of arugments. See EXAMPLES section for
sample of useage.

Output Options:

-o, --output FILE
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.)

-O, --output-BP
Output base positions on reads.

-s, --output-MQ
Output mapping quality. Equivalent to --output-extra MAPQ.

--output-QNAME
Output an extra column containing comma-separated read names. Equivalent to
--output-extra QNAME.

--output-extra STR
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.

--output-sep CHAR
Specify a different separtor 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.

--output-empty CHAR
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 *.

--reverse-del
Mark the deletions on the reverse strand with the character #, instead of the
usual *.

-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.

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 pre-calculated 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.

EXAMPLES

o Call SNPs and short INDELs:

samtools mpileup -uf ref.fa aln.bam | bcftools call -mv > var.raw.vcf
bcftools filter -s LowQual -e '%QUAL<20 || DP>100' var.raw.vcf > var.flt.vcf

The bcftools filter command marks low quality sites and sites with the read depth
exceeding a limit, which should be adjusted to about twice the average read depth
(bigger read depths usually indicate problematic regions which are often enriched for
artefacts). One may consider to add -C50 to mpileup if mapping quality is overestimated
for reads containing excessive mismatches. Applying this option usually helps BWA-short
but may not other mappers.

Individuals are identified from the SM tags in the @RG header lines. Individuals can be
pooled in one alignment file; one individual can also be separated into multiple files.
The -P option specifies that indel candidates should be collected only from read groups
with the @RG-PL tag set to ILLUMINA. Collecting indel candidates from reads sequenced
by an indel-prone technology may affect the performance of indel calling.

o Generate the consensus sequence for one diploid individual:

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

o Include customized index file as a part of arugments.

samtools mpileup [options] -X /data_folder/in1.bam [/data_folder/in2.bam [...]] /index_folder/index1.bai [/index_folder/index2.bai [...]]

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.

AUTHOR