How To Annotate And Remove Rrna/Trna From Mirna Seq Data

Question

[liran0921]

Hi All,

I am beginner at miRNA analysis. Now I am working on miRNA annotation.I have been working on it for several days but still have no idea how to do it. What I want is to annotate and remove rRNA/tRNA from my total data. Could anybody give me some guidelines or provide some resources?

I know I should blast against Rfam. And probably I should use blastn. But I haven't been able to get what I mentioned above. I would appreciate if somebody can help me on it.

Thanks!

Ran

Answer 1

[Rm]

Aligning sequences against RFAM and filtering them out that are not aligned to miRNA.

for details check these links this and this

[liran0921]

Hi Rm, thank you very much. They are quite useful, but still they just provide some guidelines. Could you provide more information about how to blast Rfam, like some command examples ?

[David Westergaard]

You can get those things from the RFarm ftp. Look under rfram_scan.

Answer 2

[Mike Axtell]

Assuming you have a reference genome for your organism, one option is not to worry about pre-filtering the miRNA seq data. After adapter trimming and alignment to the reference genome, rRNA and tRNAs that produced spurious small RNAs can be recognized easily in at least two ways --

1, based on the existing annotations of rRNA and tRNA genes in the organism, such loci can easily be found.

2, more universally, after reference-mapping, small RNA clusters that are NOT dicer-derived will often have a distinct distribution of read sizes that is NOT within the normal ~20-24 nt range. In contrast, the miRNAs you are interested in will produce clusters tightly centered around 20-22 nts.

For aligners, probably something faster then blastn is appropriate .. bowtie1 is a great aligner for small RNA-seq data.

[liran0921]

Hi Mike, thanks for your reply. I have aligned my clean reads against bovine genome. But I have no idea about how to filter those rRNA/tRNA based on existing annotations? Could you tell me which kind of program can perform this work?

Answer 3

[Jeremy Leipzig]

The tRNA thing is actually a valid fear - you will get mismappings to miRNAs unless you allow 0 mismatches. In fact there are a couple nucleotides that are added onto tRNAs that can make them not fully map to their genomic precursors but actually look even more like miRNAs.

I would download all tRNAs and use them as a blacklist: http://gtrnadb.ucsc.edu/download.html (rfam is probably even a better resource)

So before aligning to the genome you should align to the tRNAs, and remove those sequences that match perfectly.

[liran0921]

Thanks, Jeremy. Right now, I am just using tRNA from cow for filtering. So you mean that I should use the tRNA from all species?

Answer 4

[sanchezcavani]

miRNAbase is a good database for miRNA research. You may want to do homology search first. Also. you can do target prediction of your miRNAs.

[liran0921]

Thanks for your suggestion. But I think it's better if I can filter these tRNA/rRNA first.