Why little difference in RNAseq alignment when flipping forward and reverse strand with paired end reads
1
1
Entering edit mode
29 days ago

I ran bowtie2, using bulk paired RNA-seq data, with the forward and reverse strand flipped. In other words, the reverse-strand fastaq file was using the -1 argument and the forward-strand fastaq file was using the -2 arguement. Overall, there was little difference between the alignment rate (e.g. 85.38% versus 85.17%).

This struct me as odd. Can anyone explain this behavior?

Thank you!

mRNA rna-seq bowtie alignment • 621 views
ADD COMMENT
0
Entering edit mode

Did you provide any other parameter related to read orientation ?

ADD REPLY
0
Entering edit mode

I ran bowtie2, using bulk paired RNA-seq data, with the forward and reverse strand flipped.

If you know you flipped the data then re-do the analysis. It will cause issues with downstream analysis.

Aligners will try and reverse complement the reads to see which strand they map to. Did you check on the insert size stats? In your alignment files, reads would be marked as in wrong orientation (i.e. not properly paired). TLEN may also be marked as abnormally large or negative.

ADD REPLY
0
Entering edit mode
28 days ago

Bowtie doesn't know what read should go in what direction. It's aligning to genome, it will just flip things to make the alignment work.

The problem might come downstream, if it expects certain reads to align to transcripts in particular ways. You might tank your gene counts if you aligned the wrong way.

ADD COMMENT

Login before adding your answer.

Traffic: 1668 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6