Question: Untrimmed reads in small-RNA seq datasets
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gravatar for russell.stewart.j
2.3 years ago by
russell.stewart.j0 wrote:

What are the typical percentages of raw reads that remain untrimmed in Illumina HiSeq 2500 small-RNA sequencing datasets? I cannot find any papers that state both their raw read and processed read counts to get an idea. I'm working with sequencing sets from testis tissue, and leaving 16-20% of my reads untrimmed. Library prep was done with NEBNext® Small RNA Library Prep Set for Illumina®, making the adapter -AGATCGGA...3'

Thanks!

rna-seq • 728 views
ADD COMMENTlink written 2.3 years ago by russell.stewart.j0

I'm not sure if there is a 'normal', considering how much variability there can be in library creation, and other factors. Also, I've never seen NEBNext data; I've only received emails from people who work with it. So I'll ask a few questions -

1) How are you doing your trimming?
2) What's the length distribution afterward?
3) What do you expect the length distribution to be?
4) Do you expect a large percentage of your reads to be off-target?  For example, PhiX or other spike-ins.
5) Are the reads paired?
ADD REPLYlink modified 2.3 years ago • written 2.3 years ago by Brian Bushnell16k

1) How are you doing your trimming?

  • Using a custom script which trims everything after AGATCGGA. Also tried trimmomatic. Both result in 18% left over 51 nt untrimmed reads

2) What's the length distribution afterward?

  • Length distribution is as I would expect, with strong miRNA and piRNA peaks.

3) What do you expect the length distribution to be?

  • Peaks at ~21 and ~29 nt, corresponding to the above.

4) Do you expect a large percentage of your reads to be off-target? For example, PhiX or other spike-ins.

  • In retrospect, I wish we had included spike-ins. Unfortunately we didn't use any.

5) Are the reads paired?

  • I didn't realize that small-RNA datasets could have paired reads given their length, but no, not paired.

Thanks for your consideration, if you have any ideas please let me know.

ADD REPLYlink modified 2.3 years ago • written 2.3 years ago by russell.stewart.j0
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