Question: single- or pair-end small RNA seq for miRNAs?
1
gravatar for dario.veneziano
5.4 years ago by
United States
dario.veneziano10 wrote:

Hi all!

I'm in possession of a small RNA-seq dataset obtained from Illumina PE-50bp flow cell sequencing instead of SR-50bp flow cell.

Does small RNA require sequencing to be single-end only (i.e. to detect miRNAs)? Can paired-end be used as well? If yes, what tools do you suggest? Could I still make use of miRDeep2?

Thank you in advance!

 

Dario

rna-seq • 4.7k views
ADD COMMENTlink modified 5.4 years ago by h.mon31k • written 5.4 years ago by dario.veneziano10
1
gravatar for h.mon
5.4 years ago by
h.mon31k
Brazil
h.mon31k wrote:

Just merge PE1 and PE2, trim adapters (I do not know if miRDeep2 trim adapters too) and move on with your analysis - almost all your reads should merge, so should make no difference they came from PE run. BBMerge and BBDuk (from BBMap / BBTools package) do a good job at both tasks.

ADD COMMENTlink written 5.4 years ago by h.mon31k
2

One add: A simple merge of PE1 and PE2 would result in a problem and destroy your miRDeep2 run, since PE2 is the reverse complement of the sequenced molecule. Assure the you use tools like BBmerge, FLASH, PEAR, COPE, or fastq-join to do the merging step, since they assure that you get the correct strand. h.mon is saying exactly that... I just want to point out that merging the files with a linux call like 'cat PE1.fastq PE2.fastq' would fail.

And one more thing: Using paired-end sequencing for microRNA analysis does not make a lot of sense. It's a waste of time and money, since you sequence both directions and then delete one by merging them together. It might increase your quality, but the quality is normally not a problem in that length range.

ADD REPLYlink modified 5.4 years ago • written 5.4 years ago by David Langenberger9.5k

How about if I didn't merge and only considered PE1 for my analysis for example?

ADD REPLYlink written 5.4 years ago by dario.veneziano10
3

Then you waste money and information. :)

ADD REPLYlink written 5.4 years ago by David Langenberger9.5k

Now that you have both, merge them: you will increase (probably not by much, but anyway...) the overall quality of the sequences; for these short sequences, merging is a good way to tell you where are the adapters, and if PE1 and PE2 do not merge you know there is something wrong.

I will quote something I've read somewhere: "Then you waste money and information." ;-)

ADD REPLYlink modified 5.4 years ago • written 5.4 years ago by h.mon31k

Would merging take into account the paired-end nature of the reads? Would I lose info if I simply merged, or would merging give an output file as if it had been sequenced single-end? I'm concerned with the integrity of the data, since I've never heard of paired-end for small RNA sequencing...

ADD REPLYlink written 5.4 years ago by dario.veneziano10
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