Say your aim is to calculate the coverage of an RNA-seq experiment generated with long-read sequencing (so, uneven read length).
Up to now, I relied on the Lander/Waterman equation:
$$C = L*N / G$$
$C$ = final coverage
$G$ = haploid genome (or transcriptome) length
$L$ = read length
$N$ = number of reads
I have two major conceptual issues with this formula:
- $L$ is uneven in this case. Should we use the median/mean read length instead? If so, side question: if the RNA-seq experiment was executed following the size-fractionated protocol (PacBio), is it advisable to merge all the fractions prior to coverage calculation or should it be calculated separately for each fraction?
- Should $N$ consist of the mapped reads to the reference or simply the number of reads as found in the subreads whole dataset?