Hi, I am trying to use HISAT2 to align my RNA-seq data, but I am having some problems. I have 15 samples, all with stranded paired-end RNA-seq, and I am using GALAXY to process it. Now, I am in the "HISAT2" step to align it, but I think that something might be wrong, since some samples have a high percentage of reads with concordant alignment, but other samples have mostly discordant alignment.

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As you see, there is a high variation in the alignment between samples, even though I have done exactly the same process for all of them (at this point, basically cutadapt with minimum length = 20 and Quality cutoff = 20).

I am using HISAT2 in Galaxy, configurating it for "Reverse paired-end" (fr), and I have not changed anything else from the standard configuration.

So, in conclusion. Is anything wrong with my process? How could I improve it? And, if not, is it normal that different samples have this variation in alignment?

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