Deduplication

Appendix (III): Bismark Deduplication

This script is supposed to remove alignments to the same position in the genome from the Bismark mapping output (both single and paired-end SAM/BAM files), which can arise by e.g. excessive PCR amplification. If sequences align to the same genomic position but on different strands they will be scored individually.

Caution

Please note that for paired-end BAM files the deduplication script expects Read1 and Read2 to follow each other in consecutive lines! If the file has been sorted by position make sure that you resort it by read name first (e.g. using samtools sort -n)

A brief description of the Bismark deduplication and a full list of options can also be viewed by typing bismark dedup --help.

USAGE: bismark dedup [options] <filename(s)>

ARGUMENTS:

• <filenames>

A space-separated list of Bismark result files in BAM/SAM format.

OPTIONS:

• -s/--single-end

  Deduplicate single-end BAM/SAM Bismark files. Default: [AUTO-DETECT]

• -p/--paired-end

  Deduplicate paired-end BAM/SAM Bismark files. Default: [AUTO-DETECT]

• -o/--outfile [filename]

The basename of a desired output file. This basename is modified to end into .deduplicated.bam, or .multiple.deduplicated.bam in --multiple mode, for consistency reasons.

• --output_dir [path]

Output directory, either relative or absolute. Output is written to the current directory if not specified explicitly.

• --barcode

In addition to chromosome, start position and orientation this will also take a potential barcode into consideration while deduplicating. The barcode needs to be the last element of the read ID and separated by a ’:’, e.g.: MISEQ:14:000000000-A55D0:1:1101:18024:2858_1:N:0:CTCCT

• --bam

The output will be written out in BAM format. The Rust suite writes BAM directly via pure-Rust noodles, so no Samtools installation is required (the legacy --samtools_path and the .sam.gz fallback no longer apply). Default: ON.

• --sam

The output will be written out in SAM format. Default: OFF.

• --multiple

All specified input files are treated as one sample and concatenated together for deduplication. This uses Unix cat for SAM files and samtools cat for BAM files. Additional notes for BAM files: 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. By default the header is taken from the first file to be concatenated.

• --samtools_path [path]

Accepted for compatibility but ignored by the Rust suite, which does all BAM/SAM/CRAM I/O with pure-Rust noodles — no Samtools installation is required

• --version

Print version information and exit

OUTPUT:

The output is a BAM format by default, as well as a deduplication report (ending in ‘_deduplication_report.txt’)