How to use 2 paired end .sam files in featureCounts?

Question

[lauragouldgoetz]

Hello,

I split paired ends data from RNA high throughput sequencing data into two .fastq and then .sam files (using fastq-dump, then hisat2) and want to put that data into featureCounts, but the only sample code I could find in their documentation references only one file. Does anyone know how I can do this or how I should merge my two .sam files?

Thank you so much!

[h.mon]

You will have to explain more carefully what you are doing - preferably, showing the commands used. Are the two fastq files from paired-reads? If yes, they should have been mapped together.

If you "put" several files into featureCounts, it will output a table of counts, with one column per bam file. Is that what you want?

[lauragouldgoetz]

The original .sra file fileName.sra was from paired reads.

I first did: $ fastq-dump --split-files fileName.sra

Then (on the two generated files: fileName_1.fastq and fileName_2.fastq): $ hisat2 -x mm10/genome -U fileName_1.fastq -S fileName_1.sam $ hisat2 -x mm10/genome -U fileName_2.fastq -S fileName_2.sam

Now I have the two .sam files and don't know how to proceed from here to put them into featureCounts. Thanks!

[devbt15]

Yes, the hisat2 command should use both the fastq files in case of a paired end read sequencing. The resultant .bam file has to be put into featureCounts to get read counts for the features...

Answer 1

[lauragouldgoetz]

I just realized that I should have used the hisat2 command:

hisat2 -x mm10/genome -1 fileName_1.fastq -2 fileName_2.fastq -S fileName.sam

Then the resultant file goes on to featureCounts.

Thank you for your help!