Check coverage strandness
When the target enrichment is performed with highly-multiplexed PCRs (e.g. MIPs) one could be interested in checking if a region is only covered by forward or reverse reads. To allow this check, covtobed --output-strands will print two separate coverage columns:
covtobed --output-strands test/mp.bam
The output will print the coverage from forward reads in the 4th column, and coverage from reverse reads in the 5th column. The following example shows a strand bias at the beginning and the end of a chromosome:
NC_001416.1 0 2 0 0
NC_001416.1 2 13 0 1
NC_001416.1 13 14 0 2
NC_001416.1 14 18 0 3
NC_001416.1 18 21 0 4
NC_001416.1 21 31 0 5
[...]
NC_001416.1 2767 2772 5 0
NC_001416.1 2772 2774 4 0
NC_001416.1 2774 2778 3 0
NC_001416.1 2778 2796 1 0
A recently developed technique called TraDIS-Xpress and based on TraDIS (transposon sequencing), allows to screen for essential genes and to evaluate if the overexpression of a gene can improve the adaptation of a bacterium to a specific condition.
The library prepared for TraDIS-Xpress is composed by tags, DNA fragments that were adjacent to the transposon sequence in the genome, hence highlighting transposon insertion sites. The lack of coverage in a condition implies that the region with zero coverage is essential, as no insertions are tolerated.
The extended version of TraDIS, called TraDIS-Xpress, also provides an inducible promoter with the transposon, hence a strand specific peak can be interpreted as a site that is promoting the transcription of downstream DNA. For this application, covtobed can allows to easily identify regions with a strand imbalance, as shown in the picture below.
- Quadram Institute Bioscience, Press release on TraDIS-Xpress
- Muhammad Yasir, A. Keith Turner, Sarah Bastkowski, Andrew J. Page, Andrea Telatin, Minh-Duy Phan, Leigh G. Monahan, Aaron E. Darling, Mark A. Webber, Ian G. Charles, A new massively-parallel transposon mutagenesis approach comparing multiple datasets identifies novel mechanisms of action and resistance to triclosan