Question: De Novo Assembly of small RNA Reads (Illumina)
gravatar for Seq225
3.9 years ago by
Seq22590 wrote:


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

rna-seq assembly genome • 1.6k views
ADD COMMENTlink modified 3.9 years ago • written 3.9 years ago by Seq22590

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.

ADD REPLYlink modified 3.9 years ago • written 3.9 years ago by genomax83k

Tagging Brian Bushnell

ADD REPLYlink written 3.9 years ago by genomax83k
gravatar for Brian Bushnell
3.9 years ago by
Walnut Creek, USA
Brian Bushnell17k wrote:

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: in1=read1.fq in2=read2.fq out=merged.fq mininsert=17

For Tadpole: 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.

ADD COMMENTlink modified 3.9 years ago • written 3.9 years ago by Brian Bushnell17k
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