#coverage #bioinformatics #genomic #depth

bin+lib perbase

Fast and correct perbase BAM/CRAM analysis

4 releases

0.4.0 Oct 12, 2020
0.3.8 Oct 1, 2020
0.3.7 Sep 29, 2020
0.3.6 Sep 28, 2020

#44 in Science

MIT license

125KB
2K SLoC

Publish Rust API docs Crates.io Conda

A highly parallelized utility for analyzing metrics at a per-base level.

If a metric is missing, or performance is lacking. Please file a bug/feature ticket in issues.

Why?

Why perbase when so many other tools are out there? perbase leverages Rust's concurrency system to automagically parallelize over your input regions. This leads to orders of magnitude faster runtimes that scale with the compute that you have available. Additionally, perbase aims to be more accurate than other tools. EX: perbase counts DELs toward depth, bam-readcount does not, perbase does not count REF_SKIPs toward depth, sambamba does, perbase allows for mate-fixing in --fast-mode while mosdepth does not.

Installation

conda install -c bioconda perbase
# or via the rust toolchain
cargo install perbase

* Check version, conda lags behind what you will find in the release page

You can also download a binary from the releases page.

Tools

base-depth

The base-depth tool walks over every position in the BAM/CRAM file and calculates the depth, as well as the number of each nucleotide at the given position. Additionally, it counts the numbers of Ins/Dels at each position.

The output columns are as follows:

Column Description
REF The reference sequence name
POS The position on the reference sequence
REF_BASE The reference base at the position, column excluded if no reference was supplied
DEPTH The total depth at the position SUM(A, C, T, G, DEL)
A Total A nucleotides seen at this position
C Total C nucleotides seen at this position
G Total G nucleotides seen at this position
T Total T nucleotides seen at this position
N Total N nucleotides seen at this position
INS Total insertions that start at the base to the right of this position
DEL Total deletions covering this position
REF_SKIP Total reference skip operations covering this position
FAIL Total reads failing filters that covered this position (their bases were not counted toward depth)
perbase base-depth ./test/test.bam

Example output

REF     POS     REF_BASE        DEPTH   A       C       G       T       N       INS     DEL     REF_SKIP        FAIL
chr1    709636  T       16      0       0       0       16      0       0       0       0       0
chr1    709637  T       16      0       4       0       12      0       0       0       0       0
chr1    709638  A       16      16      0       0       0       0       0       0       0       0
chr1    709639  G       16      0       0       16      0       0       0       0       0       0
chr1    709640  A       16      16      0       0       0       0       0       0       0       0
chr1    709641  A       16      16      0       0       0       0       0       0       0       0
chr1    709642  G       16      0       0       16      0       0       0       0       0       0
chr1    709643  G       16      0       0       16      0       0       0       0       0       0
chr1    709644  T       16      0       0       0       16      0       0       0       0       0
chr1    709645  G       16      0       0       16      0       0       0       0       0       0

If the --mate-fix flag is passed, each position will first check if there are any mate overlaps and choose the mate with the hightest MAPQ, breaking ties by choosing the first mate that passes filters. Mates that are discarded are not counted toward FAIL or DEPTH.

If the --reference-fasta is supplied, the REF_BASE field will be filled in. The reference must be indexed an match the BAM/CRAM header of the input.

The output can be compressed and indexed as follows:

perbase base-depth ./test/test.bam | bgzip > output.tsv.gz
tabix -S 1 -s 1 -b 2 -e 2 ./output.tsv.gz
# Query all positions overlapping region
tabix output.tsv.gz chr1:5-10

Usage:

perbase-base-depth 0.3.9-alpha.0
Seth Stadick <sstadick@gmail.com>
Calculate the depth at each base, per-nucleotide

USAGE:
    perbase base-depth [FLAGS] [OPTIONS] <reads>

FLAGS:
    -h, --help         Prints help information
    -m, --mate-fix     Fix overlapping mates counts, see docs for full details
    -V, --version      Prints version information
    -z, --zero-base    Output positions as 0-based instead of 1-based

OPTIONS:
    -b, --bed-file <bed-file>                A BED file containing regions of interest. If specified, only bases from
                                             the given regions will be reported on
    -c, --chunksize <chunksize>              The ideal number of basepairs each worker receives. Total bp in memory at
                                             one time is (threads - 2) * chunksize
    -F, --exclude-flags <exclude-flags>      SAM flags to exclude, recommended 3848 [default: 0]
    -f, --include-flags <include-flags>      SAM flags to include [default: 0]
    -q, --min-mapq <min-mapq>                Minimum MAPQ for a read to count toward depth [default: 0]
    -o, --output <output>                    Output path, defaults to stdout
        --ref-cache-size <ref-cache-size>    Number of Reference Sequences to hold in memory at one time. Smaller will
                                             decrease mem usage [default: 10]
    -r, --ref-fasta <ref-fasta>              Indexed reference fasta, set if using CRAM
    -t, --threads <threads>                  The number of threads to use [default: 16]

ARGS:
    <reads>    Input indexed BAM/CRAM to analyze

only-depth

The only-depth tool walks over the input BAM/CRAM file and calculates the depth over all positions specified by either a BED file or in the BAM/CRAM header. Adjacent positions that have the same depth will be merged together to form a non-inclusive range (see example output).

There are two distinct modes that only-depth can run in, gated by the --fast-mode flag. When running in fast-mode, only depth over the area a read covers is only determined by the reads start and end postions, and no cigar related info is taken into account. --mate-fix may still be used in this mode, and areas where mates overlap will not be counted twice.

Without the --fast-mode flag, the depth at each position is determined in a manner similar to base-depth where DEL will count toward depth, but REF_SKIP will not. Additionally, any reads that fail the --exclude-flags will not be counted toward depth. Lastly, --mate-fix can be applied to avoid counting regions twice where mates may overlap.

Regarding mate fixes, perbase will will "fixes" based only on the counted regions in a read. For example, if you have a read that goes from "chr1:10-1000" with a CIGAR of "25M974N1M", and the mate alignes nicely at "chr1:45-70" with CIGAR "25M", the mate will count toward the depth over "chr1:45-74". This is in contrast to other tools that will reject the mate even though it overlaps a region of R1 that is not counted toward depth.

For the fastest possible output, use only-depth --fast-mode. --mate-fix is more computational.

Note that it is possible that two adjacent positions may not merge if they fall at a --chunksize boundary. If this is an issue you can set the --chunksize to the size of the largest contig in question. At a future date this may be fixed or a post processing tool may be provided to fix it. For most use cases this should not be a problem. Additionally, you can pipe into merge-adjcent which will fix it as well. EX: perbase only-depth -m file.bam | perbase merge-adjacent > out.tsv.

Example output of perbase only-depth --mate-fix --zero-base ./test/test.bam:

REF     POS     END     DEPTH
chr2    0       4       1
chr2    4       9       2
chr2    9       12      3
chr2    12      14      2
chr2    14      17      3
chr2    17      19      4
chr2    19      23      5
chr2    23      34      4
chr2    34      39      3
chr2    39      49      1
chr2    49      54      2
chr2    54      64      3
chr2    64      74      4
chr2    74      79      3
chr2    79      84      2
chr2    84      89      1

Usage:

Calculate the only the depth at each base

USAGE:
    perbase only-depth [FLAGS] [OPTIONS] <reads>

FLAGS:
    -x, --fast-mode    Calculate depth based only on read starts/stops, see docs for full details
    -h, --help         Prints help information
    -m, --mate-fix     Fix overlapping mates counts, see docs for full details
    -n, --no-merge     Skip merging adjacent bases that have the same depth
    -V, --version      Prints version information
    -z, --zero-base    Output positions as 0-based instead of 1-based

OPTIONS:
    -b, --bed-file <bed-file>              A BED file containing regions of interest. If specified, only bases from the
                                           given regions will be reported on
    -c, --chunksize <chunksize>            The ideal number of basepairs each worker receives. Total bp in memory at one
                                           time is (threads - 2) * chunksize
    -F, --exclude-flags <exclude-flags>    SAM flags to exclude, recommended 3848 [default: 0]
    -f, --include-flags <include-flags>    SAM flags to include [default: 0]
    -q, --min-mapq <min-mapq>              Minimum MAPQ for a read to count toward depth [default: 0]
    -o, --output <output>                  Output path, defaults to stdout
    -r, --ref-fasta <ref-fasta>            Indexed reference fasta, set if using CRAM
    -t, --threads <threads>                The number of threads to use [default: 16]

ARGS:
    <reads>    Input indexed BAM/CRAM to analyze

merge-adjacent

merge-adjacent is a utility to merege overlapping regions in a BED-like file.

It will take a file with four columns and no header as long as the colums are like:

<contig>\t<start>\t<stop>\t<depth>\n

Or it can take files with three columsn with headers that are like

<REF|chrom>\t<POS|chromStart>\t<END|chromEnd>\t<DEPTH|COV>

The END|chromEnd column is optional.

Merge adjacent intervals that have the same depth. Input must be sorted like: `sort -k1,1 -k2,2n in.bed > in.sorted.bed`

Generally accepts any file with no header tha is <chrom>\t<start>\t<stop>\t<depth>. The <stop> is optional. See
documentation for explaination of headers that are accepted.

USAGE:
    perbase merge-adjacent [FLAGS] [OPTIONS] [in-file]

FLAGS:
    -h, --help
            Prints help information

    -n, --no-header
            Indicate if the input file does not have a header

    -V, --version
            Prints version information


OPTIONS:
    -o, --output <output>
            The output location, defaults to STDOUT


ARGS:
    <in-file>
            Input bed-like file, defaults to STDIN

EX:

perbase only-depth indexed.bam | perbase merge-adjacent > out.tsv

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