Question: Small RNA data analysis
1
gravatar for jkkbuddika
5 weeks ago by
jkkbuddika160
Bloomington, USA
jkkbuddika160 wrote:

Hi,

I am in the process of writing a script to automate small RNA-seq data analysis (18-30nt read length) to identify miRNAs and other types of small RNAs. Based on my reading this is what I came up with.

  1. Trim adaptors with Cutadapt.
  2. Alignment using Bowtie v1 (old version, not bowtie2).
  3. Count miRNAs using featureCounts (in the case of miRNA).

Does this look good? If not what other steps are recommended?

Thanks in advance,

KB

rna-seq alignment • 161 views
ADD COMMENTlink modified 2 days ago by Biostar ♦♦ 20 • written 5 weeks ago by jkkbuddika160
1

Some miRNA kits require kit specific data handling (e.g. they may use a 3' adapter that is directly ligated to ends of miRNA and needs to be specifically removed, only reads containing that adapter may be real miRNA). I would definitely check on that possibility.

ADD REPLYlink written 5 weeks ago by genomax91k

Thanks a lot. Will look into that. Do you think Bowtie v1 is the best choice for alignment, over let's say STAR or bowtie2?

ADD REPLYlink written 5 weeks ago by jkkbuddika160

Yes. You want to do ungapped alignments due to the small sizes of miRNA.

ADD REPLYlink written 5 weeks ago by genomax91k

Awesome. Thanks a lot for all the suggestions.

ADD REPLYlink written 5 weeks ago by jkkbuddika160

@genomax, I haven't tried bowtie yet. If needed, what parameters would you recommend changing for alignment? I was thinking first running in the default mode and see.

ADD REPLYlink written 5 weeks ago by jkkbuddika160
1

There is a study looking at different aligners for performing miRNA alignment which could be of use for you (table 3 discusses parameters used): Evaluation of microRNA alignment techniques

ADD REPLYlink written 5 weeks ago by newbio17240

That's helpful. Thanks. I ran with default settings and the alignment rate looked pretty good!

ADD REPLYlink written 5 weeks ago by jkkbuddika160
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