I got some RNASeq fastq data from a customer, and he told me the samples were mainly from human cell lines but may have some contamination with mouse cells. My question is whether I should align those sequences against both human genome reference and mouse genome reference or just humna's. Any suggestions?
- First align all the samples to the human genome.
- Then align the un-mapped reads to the mouse genome.
If you get a large portion of (un-mapped) reads mapping to mouse, then it's very likely the sample was contaminated.
First subset the files (seqtk) and then use fastq_screen to get an idea what the contamination rate is. I've found it useful to only pay close attention to the "single alignment in a single organism" (or whatever that's called) category, since the others are more an indicator of sequence complexity. I happen to do this with all sequencing runs produced at our institute, since it immediately allows us to flag problematic samples (anything over 0.5% off-species unique alignment is a problem).
Ideally you won't have much contamination and if you do you can just exclude the sample. If you can't exclude the sample, then you'll need to simultaneously align to both genomes (get one from Ensembl and the other from UCSC, so the chromosome names differ, and then concatenate them). Align against the concatenated genome and then extract only the human reads with some meaningful MAPQ threshold. One can get more elegant with this, but that should suffice 99.9% of the time.