3.2 years ago by
WCIP | Glasgow | UK
Can I use either single reads or fragments (i.e. paired reads) to calculate RPKM?
What definitely you do not want to do is to count both read 1 and read 2. Counting fragments (i.e. both mates mapping to the same gene in correct orientation?) should be more appropriate but in practice I think counting only read 1s should be the same.
If I only want to calculate RPKM for mitochondrial genes, should I use A or B during calculation?
I guess it depends on whether you are interested in the concentration of a mitochondrial gene within mitochondrial genes or among all the genes. In the first case use mitochondrial reads otherwise use everything.
For example, in one sample you have 1M reads in the genome, 10k reads on chrM and 100 reads on gene X on chrM. In another sample you have 1M reads in the genome, 1k reads on chrM and 100 reads on gene X. In this case the concentration of X is pretty much the same in the two samples, genomewide. But relative to the mitochondrial genome the second sample is much richer in gene X (100/10k vs 100/1k).