This tutorial teaches how to run a quick sequencing depth and coverage check for one or more reference sequences.
1. What is Sequencing Depth and Coverage?
Sequencing depth refers to the number of times a particular DNA or RNA sequence has been read during a sequencing experiment. This number is important because the more times a sequence is read, the more accurate the resulting data will be. The sequencing depth also helps to determine the coverage, which is the fraction of the target genome or transcriptome that has been sequenced to a particular depth. In other words, coverage measures how well the genome or transcriptome has been sampled.
For example, a coverage of 20x means that on average, each base in the target genome or transcriptome has been sequenced 20 times. The goal is usually to have a high coverage, meaning that the target genome or transcriptome has been sequenced in great detail and with high accuracy. The coverage required for a particular experiment depends on the specifics of the study, but generally, a coverage of 30x to 50x is considered sufficient for many applications.
2. Sequencing Depth and Coverage Check Using Samtools
In bioinformatics, very often, checking for the coverage and depth of a given reference sequence is required.
Samtools introduced on version 1.10 a “coverage” as a sub-command that can be used to process BAM files and return the coverage and other valuable metrics (listed below) for each of the reference sequences.
The tool usage is pretty simple:
$ samtools coverage BAM_file -o OUTPUT
The call returns the following columns:
- rname: Reference name / chromosome
- startpos: Start position
- endpos: End position (or sequence length)
- numreads: Number reads aligned to the region (after filtering)
- covbases: Number of covered bases with depth >= 1
- coverage: Proportion of covered bases [0..1]
- meandepth: Mean depth of coverage
- meanbaseq: Mean baseQ in covered region
- meanmapq: Mean mapQ of selected reads
For more information on the sub-command and filters, please check the help with “samtools coverage -h.”