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How to handle paired and unpaired reads in RNAseq pipeline

Hello everyone. I am currently designing a pipeline which takes in RNA sequencing data and ideally will do a lot of the general pipeline operations (trim -> align -> differential expression). After trimming (which I did using trimmomatic), I end up with both paired end fastq files and (much smaller) unpaired fastq files.

I was wondering what the best way to proceed with these files is. So far, I've been trying to handle them separately. I aligned them separately using STAR and used RSEM separately for quantification. At this point, would you recommend that I merge the counts? is there an easy way to do this? Or is my whole thought process wrong. I'd appreciate any suggestions

rnaseq
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updated by ATpoint ATpoint • written by mohamadelian1996 • mohamadelian1996

1 answer

You should just not use the unpaired FASTQ files… it’s like 1% of your reads; I don’t know why you’d care about preserving them. It adds unneeded complexity to pipelines.

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written by dsull dsull

It is not standard practice to keep them, and they don't suggest that you should keep them (they're giving suggestions if you already go through the hassle of keeping them, in which case, for RSEM, I'd say merge the BAM files then run RSEM).

I would discard them. There's no need to keep that extra 1% of your reads unless you have some good reason to do so. You're already going to have literally millions (maybe billions) of reads in your dataset; I don't know what good an extra 1% of reads will do you.

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written by dsull dsull

Seconding all this. Unpairead reads after trimming are a neglectible fraction, and non-uniform processing rather adds uncertainty and adding value. Keep it simple and toss them.

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written by ATpoint ATpoint

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