Paired end reads not properly aligning in Rsubread align

Question

[Bonita]

Hi all,

I am trying to use Rsubread to align paired end RNAseq data to a mouse genome index that I built with the bulidindex function in Rsubread. I then used the command below to try to align my sequences to the index.

align(index="GRCm39", readfile1=allRead1files, readfile2 = allRead2files)

allReadfiles1 and allRead2files are vectors of the file names that I created. I had them written into a file and they are the correct file names in the same order. My understanding is that this will feed line 1 fro allRead1files and line 1 from allRead2files into the command, then line 2, ect. If that is the case it should be the correct files being used.

BUT, as you can see below the paired end reads are not aligning to the index properly. It seems to be an issue with the strandedness?

What could be causing this? Could I still use the BAM files for further analysis?

Thanks in advance.

Answer 1

[Bonita]

If anyone else is having the same problem it is because my sequences were both in the forward direction in the files. Changing the command to:

align(index="GRCm39", readfile1=allRead1files, readfile2 = allRead2files, PE_orientation="ff")

fixed the problem. The default setting is "fr".

[suhanyavp]

Thank you for this solution. I ran into the same problem and have now found the solution. I would like to know if you have used featurecounts after the align step and if so what have you mentioned in the Strandspecific option ? (0 or 2)