samtools-stats(1)

NAME
samtools-stats - produces comprehensive statistics from alignment file

SYNOPSIS

samtools stats [options] in.sam|in.bam|in.cram [region...]

DESCRIPTION

samtools stats collects statistics from BAM files and outputs in a text format. The output can be visualized graphically using plot-bamstats.

A summary of output sections is listed below, followed by more detailed descriptions.

CHK | Checksum
SN | Summary numbers
FFQ | First fragment qualities
LFQ | Last fragment qualities
GCF | GC content of first fragments
GCL | GC content of last fragments
GCC | ACGT content per cycle
GCT | ACGT content per cycle, read oriented
FBC | ACGT content per cycle for first fragments only
FTC | ACGT raw counters for first fragments
LBC | ACGT content per cycle for last fragments only
LTC | ACGT raw counters for last fragments
BCC | ACGT content per cycle for BC barcode
CRC | ACGT content per cycle for CR barcode
OXC | ACGT content per cycle for OX barcode
RXC | ACGT content per cycle for RX barcode
MPC | Mismatch distribution per cycle
QTQ | Quality distribution for BC barcode
CYQ | Quality distribution for CR barcode
BZQ | Quality distribution for OX barcode
QXQ | Quality distribution for RX barcode
IS | Insert sizes
RL | Read lengths
FRL | Read lengths for first fragments only
LRL | Read lengths for last fragments only
MAPQ | Mapping qualities
ID | Indel size distribution
IC | Indels per cycle
COV | Coverage (depth) distribution
GCD | GC-depth

The "cycle" terminology used here originates from the Illumina instruments, but it is interpreted more generally as the Nth base reported in the original read orientation (starting from 1).

Not all sections will be reported as some depend on the data being coordinate sorted while others are only present when specific barcode tags are in use.

Some of the statistics are collected for “first” or “last” fragments. Records are put into these categories using the PAIRED (0x1), READ1 (0x40) and READ2 (0x80) flag bits, as follows:

•   Unpaired reads (i.e. PAIRED is not set) are all “first” fragments. For these records, the READ1 and READ2 flags are ignored.

•   Reads where PAIRED and READ1 are set, and READ2 is not set are “first” fragments.

•   Reads where PAIRED and READ2 are set, and READ1 is not set are “last” fragments.

•   Reads where PAIRED is set and either both READ1 and READ2 are set or neither is set are not counted in either category.

Information on the meaning of the flags is given in the SAM specification document <https://samtools.github.io/hts-specs/SAMv1.pdf>.

The CHK row contains distinct CRC32 checksums of read names, sequences and quality values. The checksums are computed per alignment record and summed, meaning the checksum does not change if the input file has the sort-order changed.

NOTE: Checksum calculation for quality values was modified and quality checksum value will be different from that generated using versions up to 1.21.

The SN section contains a series of counts, percentages, and averages, in a similar style to samtools flagstat, but more comprehensive.

    raw total sequences - total number of reads in a file, excluding supplementary and secondary reads. Same number reported by samtools view -c -F 0x900.

    filtered sequences - number of discarded reads when using -f or -F option.

    sequences - number of processed reads.

    is sorted - flag indicating whether the file is coordinate sorted (1) or not (0).

    1st fragments - number of first fragment reads (flags 0x01 not set; or flags 0x01 and 0x40 set, 0x80 not set).

    last fragments - number of last fragment reads (flags 0x01 and 0x80 set, 0x40 not set).

    reads mapped - number of reads, paired or single, that are mapped (flag 0x4 or 0x8 not set).

    reads mapped and paired - number of mapped paired reads (flag 0x1 is set and flags 0x4 and 0x8 are not set).

    reads unmapped - number of unmapped reads (flag 0x4 is set).

    reads properly paired - number of mapped paired reads with flag 0x2 set.

    paired - number of paired reads, mapped or unmapped, that are neither secondary nor supplementary (flag 0x1 is set and flags 0x100 (256) and 0x800 (2048) are not set).

    reads duplicated - number of duplicate reads (flag 0x400 (1024) is set).

    reads MQ0 - number of mapped reads with mapping quality 0.

    reads QC failed - number of reads that failed the quality checks (flag 0x200 (512) is set).

    non-primary alignments - number of secondary reads (flag 0x100 (256) set).

    supplementary alignments - number of supplementary reads (flag 0x800 (2048) set).

    total length - number of processed bases from reads that are neither secondary nor supplementary (flags 0x100 (256) and 0x800 (2048) are not set).

    total first fragment length - number of processed bases that belong to first fragments.

    total last fragment length - number of processed bases that belong to last fragments.

    bases mapped - number of processed bases that belong to reads mapped.

    bases mapped (cigar) - number of mapped bases filtered by the CIGAR string corresponding to the read they belong to. Only alignment matches(M), inserts(I), sequence matches(=) and sequence mismatches(X) are counted.

    bases trimmed - number of bases trimmed by bwa, that belong to non secondary and non supplementary reads. Enabled by -q option.

    bases duplicated - number of bases that belong to reads duplicated.

    mismatches - number of mismatched bases, as reported by the NM tag associated with a read, if present.

    error rate - ratio between mismatches and bases mapped (cigar).

    average length - ratio between total length and sequences.

    average first fragment length - ratio between total first fragment length and 1st fragments.

    average last fragment length - ratio between total last fragment length and last fragments.

    maximum length - length of the longest read (includes hard-clipped bases).

    maximum first fragment length - length of the longest first fragment read (includes hard-clipped bases).

    maximum last fragment length - length of the longest last fragment read (includes hard-clipped bases).

    average quality - ratio between the sum of base qualities and total length.

    insert size average - the average absolute template length for paired and mapped reads.

    insert size standard deviation - standard deviation for the average template length distribution.

    inward oriented pairs - number of paired reads with flag 0x40 (64) set and flag 0x10 (16) not set or with flag 0x80 (128) set and flag 0x10 (16) set.

    outward oriented pairs - number of paired reads with flag 0x40 (64) set and flag 0x10 (16) set or with flag 0x80 (128) set and flag 0x10 (16) not set.

    pairs with other orientation - number of paired reads that don't fall in any of the above two categories.

    pairs on different chromosomes - number of pairs where one read is on one chromosome and the pair read is on a different chromosome.

    percentage of properly paired reads - percentage of reads properly paired out of sequences.

    bases inside the target - number of bases inside the target region(s) (when a target file is specified with -t option).

    percentage of target genome with coverage > VAL - percentage of target bases with a coverage larger than VAL. By default, VAL is 0, but a custom value can be supplied by the user with -g option.

The FFQ and LFQ sections report the quality distribution per first/last fragment and per cycle number. They have one row per cycle (reported as the first column after the FFQ/LFQ key) with remaining columns being the observed integer counts per quality value, starting at quality 0 in the left-most row and ending at the largest observed quality. Thus each row forms its own quality distribution and any cycle specific quality artefacts can be observed.

GCF and GCL report the total GC content of each fragment, separated into first and last fragments. The columns show the GC percentile (between 0 and 100) and an integer count of fragments at that percentile.

GCC, FBC and LBC report the nucleotide content per cycle either combined (GCC) or split into first (FBC) and last (LBC) fragments. The columns are cycle number (integer), and percentage counts for A, C, G, T, N and other (typically containing ambiguity codes) normalised against the total counts of A, C, G and T only (excluding N and other).

GCT offers a similar report to GCC, but whereas GCC counts nucleotides as they appear in the SAM output (in reference orientation), GCT takes into account whether a nucleotide belongs to a reverse complemented read and counts it in the original read orientation. If there are no reverse complemented reads in a file, the GCC and GCT reports will be identical.

FTC and LTC report the total numbers of nucleotides for first and last fragments, respectively. The columns are the raw counters for A, C, G, T and N bases.

MPC reports the number of mismatches per cycle and per quality value. The MPC statistics are only included when a reference is specified via the -r option. There is one row per cycle number. Each row includes the cycle number, the number of N bases (not counted in the per-qual columns), followed by one column per quality value (starting at zero and incrementing by one each time) listing the number of non-N mismatches with that quality. A mismatch is defined as an ACGT sequence base mismatching an ACGT reference base. Ambiguity codes are ignored (except for sequence N as mentioned above, which is counted even when the reference is also N).

BCC, CRC, OXC and RXC are the barcode equivalent of GCC, showing nucleotide content for the barcode tags BC, CR, OX and RX respectively. Their quality values distributions are in the QTQ, CYQ, BZQ and QXQ sections, corresponding to the BC/QT, CR/CY, OX/BZ and RX/QX SAM format sequence/quality tags. These quality value distributions follow the same format used in the FFQ and LFQ sections. All these section names are followed by a number (1 or 2), indicating that the stats figures below them correspond to the first or second barcode (in the case of dual indexing). Thus, these sections will appear as BCC1, CRC1, OXC1 and RXC1, accompanied by their quality correspondents QTQ1, CYQ1, BZQ1 and QXQ1. If a separator is present in the barcode sequence (usually a hyphen), indicating dual indexing, then sections ending in "2" will also be reported to show the second tag statistics (e.g. both BCC1 and BCC2 are present).

IS reports insert size distributions with one row per size, reported in the first column, with subsequent columns for the frequency of total pairs, inward oriented pairs, outward orient pairs and other orientation pairs. The -i option specifies the maximum insert size reported.

RL reports the distribution for all read lengths, with one row per observed length (up to the maximum specified by the -l option). Columns are read length and frequency. FRL and LRL contains the same information separated into first and last fragments.

MAPQ reports the mapping qualities for the mapped reads, ignoring the duplicates, supplementary, secondary and failing quality reads.

ID reports the distribution of indel sizes, with one row per observed size. The columns are size, frequency of insertions at that size and frequency of deletions at that size.

IC reports the frequency of indels occurring per cycle, broken down by both insertion / deletion and by first / last read. Note for multi-base indels this only counts the first base location. Columns are cycle, number of insertions in first fragments, number of insertions in last fragments, number of deletions in first fragments, and number of deletions in last fragments.

COV reports a distribution of the alignment depth per covered reference site. For example an average depth of 50 would ideally result in a normal distribution centred on 50, but the presence of repeats or copy-number variation may reveal multiple peaks at approximate multiples of 50. The first column is an inclusive coverage range in the form of [min-max]. The next columns are a repeat of the maximum portion of the depth range (now as a single integer) and the frequency that depth range was observed. The minimum, maximum and range step size are controlled by the -c option. Depths above and below the minimum and maximum are reported with ranges [<min] and [max<].

GCD reports the GC content of the reference data aligned against per alignment record, with one row per observed GC percentage reported as the first column and sorted on this column. The second column is a total sequence percentile, as a running total (ending at 100%). The first and second columns may be used to produce a simple distribution of GC content. Subsequent columns list the coverage depth at 10th, 25th, 50th, 75th and 90th GC percentiles for this specific GC percentage, revealing any GC bias in mapping. These columns are averaged depths, so are floating point with no maximum value.

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 requirement) [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 [-1]

-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 calculation). []

-S, --split TAG
    In addition to the complete statistics, also output categorised statistics 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>.bamstat, 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.

-p, --remove-overlaps
    Remove overlaps of paired-end reads from coverage and base count computations.

-g, --cov-threshold INT
    Only bases with coverage above this value will be included in the target percentage computation [0]

-X  If this option is set, it will allows user to specify customized index file location(s) if the data folder does not contain any index file. Example usage: samtools stats [options] -X /data_folder/data.bam /index_folder/data.bai chrM:1-10

-@, --threads INT
    Number of input/output compression threads to use in addition to main thread [0].

AUTHOR

Written by Petr Danacek with major modifications by Nicholas Clarke, Martin Pollard, Josh Randall, and Valeriu Ohan, all from the Sanger Institute.

SEE ALSO
samtools(1), samtools-flagstat(1), samtools-idxstats(1)

Samtools website: <http://www.htslib.org/>