I have seen the question briefly discussed in specific contexts, but as it seems like there is no consensus between different tools on this issue I think it would be useful to report here what the best practice is.
So I have Illumina MiSeq paired-end reads. Let's say a region is covered by a single pair of reads (one forward/one reverse), should the coverage in the overlapping region reported as 1x or 2x? What are the arguments for each?
Here is an illustration here: https://bioinformatics.stackexchange.com/questions/5427/double-counting-coverage-of-overlapped-read-pairs/5473
•
written
by
francois •
1 answer
According to the "Sequencing Coverage Calculation Methods for Human Whole-Genome Sequencing" Illumina technote, sequencing coverage is “the average coverage of unique reads across the non-N portion of the human genome.” This definition excludes not only counting overlapping regions 2x (they should be counted 1x), it also excludes PCR and optical duplicates from the calculation as well.
I think this is the most sensible definition, as we use the coverage information to know if we likely have enough information to perform downstream analyses, and sequencing the same molecule over and over again does not add independent information. That said, Illumina itself ignores the above definition, as the footnotes from table 1 indicate:
b. The Isaac WGS App v1.0 excludes overlapping bases from only
one read (ie, overlapping bases are counted one time).
c. The BWA WGS App does not exclude overlapping bases from
either read (ie, overlapping bases are counted twice).
Log in to answer this question.
-
Diffbind or DESEQ2 Chipseq data
written by Irene •
-
Read count vs Depth
written by Mary •
-
Best practices for unstranded sequences in featureCounts
written by Enrique •
-
Tutorial: Yes .. BBMap can do that! - Part II randomreads (read simulation), demuxbyname/filterbyna…
written by GenoMax
-
Strange coverage of mtDNA - need help interpreting MiSeq results
written by tatiatcg •
-
How to predict non-sense mediated decay from Illumina reads?
written by francois •
-
Depth of Coverage without Read Overlap
written by drkennetz
-
Understanding ATAC-seq data
written by Friederike
-
Tool: Tools to merge overlapping paired-end reads
written by Abdul Rafay Khan
-
Tool: RNA-Seq paired-end sequencing tophat outputted SAM file: single line or double lines?
written by lliu.hsph •
Two reads from a pair are sequencing/sampling a single unique fragment.
That said the fragment was sequenced twice to generate the two reads so if you think of coverage as number of times a region was sequenced then that would be 2.