How to properly map 4 lanes for differential expression using BWA?

Question

[satshil.r]

Hey guys,

I have 8 samples, each sample having 4 lanes. How do I go about mapping the sample to my reference when there's multiple lanes?

I'm not sure how cating the 4 lanes together would work out for counts. Here's my thought process:

If I cat the 4 lanes together before mapping (BWA), and then run BWA mem, won't that give me 4x the expression/counts since the mapping consists of 4 duplicate reads?

If this is actually what would happen, or do I have the process all wrong?

How would you properly map/align these 4-lane reads?

Answer 1

[Devon Ryan]

Splitting a library over 4 lanes doesn't result in 4 copies of every read, no one would spend the extra money to run the additional 3 lanes then. Just concatenate the files and map them, though I wouldn't recommend bwa for RNAseq (it's not splice aware, try STAR or hisat or even BBMap, which quite flexible).