Question: How to use 2 paired end .sam files in featureCounts?
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gravatar for lauragouldgoetz
21 months ago by
lauragouldgoetz10 wrote:

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!

sequencing rna-seq alignment • 991 views
ADD COMMENTlink modified 17 months ago by devbt1510 • written 21 months ago by lauragouldgoetz10
1

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?

ADD REPLYlink written 21 months ago by h.mon24k

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!

ADD REPLYlink written 21 months ago by lauragouldgoetz10
1

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...

ADD REPLYlink written 17 months ago by devbt1510
1
gravatar for lauragouldgoetz
21 months ago by
lauragouldgoetz10 wrote:

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!

ADD COMMENTlink modified 21 months ago by Istvan Albert ♦♦ 80k • written 21 months ago by lauragouldgoetz10
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