Question: featureCounts for aligned reads
0
gravatar for rsafavi
2.1 years ago by
rsafavi50
rsafavi50 wrote:

Hi everyone,

I have paired-end RNAseq data, and I used STAR aligner to do alignment, so basically for each paired-end fastq files I ended-up creating one alignment file (bam). Now I would like to create a count matrix to do DE analysis. I would like to use featureCounts tool. I was wondering what are the optimal flags to use? I am not sure if I have to use -p flag, since my reads are already aligned to the reference.

Thanks!

ADD COMMENTlink modified 2.1 years ago by genomax87k • written 2.1 years ago by rsafavi50
2
gravatar for genomax
2.1 years ago by
genomax87k
United States
genomax87k wrote:

You should use -p flag so as to count fragments. Don't use -M flag since you don't want to count multi-mapped reads. Use -t to count at exon level but summarize the counts per gene identifier using -g. If your library was stranded you will want to use appropriate -s option (most likely 2). Provide all BAM files (in the order that you want the columns to be in) in a single command to get read matrix. Your BAM files can be sorted or unsorted. If they are unsorted then featureCounts will sort them on the fly.

Here is a summary of possible command lines.

In future, you could use STAR itself to generate the counts.

ADD COMMENTlink modified 2.1 years ago • written 2.1 years ago by genomax87k

Works perfectly thank you very much!

ADD REPLYlink written 2.1 years ago by rsafavi50

One more question, is having this warning normal? I get it with -p option

WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs).

ADD REPLYlink written 2.1 years ago by rsafavi50

Were your BAM files sorted? featureCounts should sort the files as needed. You could always pre-sort them with samtools.

ADD REPLYlink written 2.1 years ago by genomax87k

should allowMultiOverlap be used or not?

ADD REPLYlink written 11 months ago by Arindam Ghosh300
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1735 users visited in the last hour