De Novo Assembly of small RNA Reads (Illumina)

Question

[Seq225]

Hello,

I want to de novo assembly of small RNA reads generated from Illumina sequencing (final lengths after clipping and trimming are 18-32nt).

Is there any pipeline available?

I tried Velvet. I used k-mer of 13 to 19 but could not assemble the reads. Assembled reads were maximum 70nt. My library is very deep and outstandingly overlapping (as most are piRNA). I really want to get the whole transcript (assembled).

Is any help out here?

Thanks -Juulluu21

[GenoMax]

You may be able to do this using BBMerge/Tadpole from BBMap. I am going to tag @Brian (author of BBMap) so he can provide some authoritative advice.

[GenoMax]

Tagging Brian Bushnell

Answer 1

[Brian Bushnell]

I had never heard of piRNAs before, but Wikipedia claims they are 26-31 bp long, so, your sequences appear to be in the target range. If you assembled something and it ended up 70bp long or longer, I assume that would be a misassembly, or that the Wikipedia article is wrong, or else that it's not a piRNA.

At any rate, you can certainly get small RNA sequences from BBMerge or Tadpole, with the correct parameters. For BBMerge:

bbmerge.sh in1=read1.fq in2=read2.fq out=merged.fq mininsert=17

For Tadpole:

tadpole.sh in1=read1.fq in2=read2.fq out=contigs.fa k=17 mincontig=17 mincov=10 mincr=5

It's pretty hard to get useful assemblies with k=17, though, so certainly use a longer value representing whatever you expect to be the shortest RNA you are interested in. Tadpole may or may not do better than Velvet - normally, it's better for highly variable coverage, but not otherwise.