Question: low % of read map to mirase mature miRNAs
1
gravatar for prp291
4.3 years ago by
prp29150
United States
prp29150 wrote:

I have some small RNA-seq data and I want to predict the miRNA sequence. Genome sequences is available for my organism. After preprocessing like adapter removal, redundant reads removal I choose only 18-24 bp long reads for further analysis. I align this reads with  genome and chose only those reads which align with genome for next step. I align reads with RFAM, plant repeats and mRNA sequence of my genome to discard any contaminations.  Final reads (which are not aligned with  RFAM, plant repeats and mRNA )  are aligned with mature miRNA sequence obtained from miRBASE database. but bowtie result comes like that

 

 2562148 (100.00%) were unpaired; of these:
    2560555 (99.94%) aligned 0 times
    264 (0.01%) aligned exactly 1 time
    1329 (0.05%) aligned >1 times
0.06% overall alignment rate

 

is it OK to get such a low number of reads?? I use this command for bowtie alignment

bowtie2 -N 0 --un unaligned/unaligned_mirbase_mature.fasta  -x mature_mirna/mirbase_mature -f aligned/aligned_to_genome.fasta

 

 

 

Thanks

 

bowtie rna-seq • 1.9k views
ADD COMMENTlink modified 4.3 years ago by David Langenberger9.1k • written 4.3 years ago by prp29150

What is the protocol for library preparation? Did it involve a poly-A selection?

ADD REPLYlink written 4.3 years ago by h.mon27k

yes it includes that. Thanks

ADD REPLYlink written 4.3 years ago by prp29150

Do mature microRNAs have a poly-A tail?

ADD REPLYlink written 4.3 years ago by David Langenberger9.1k

It is not clear to me he is using an appropriate library preparation protocol, but I have little experience with small RNA. And no, I believe mature microRNAs do not have poly-A tails.

ADD REPLYlink written 4.3 years ago by h.mon27k

Why would you remove redundant reads? That's kinda counterproductive. I highly recommend you to first read publications about small RNA-Seq and microRNAs, before performing any further analysis. 

My tip: Think about the protocol and think about the molecules you sequenced... would you expect redundancy???

ADD REPLYlink written 4.3 years ago by David Langenberger9.1k

He removed redundant reads, thats explains the low result :) he just discard them all and leave one read for each miR

ADD REPLYlink modified 4.3 years ago • written 4.3 years ago by manekineko130

Be careful with discarding repeats and mRNA hits as some miRNA have transposon or exon origin an you will lose those.

ADD REPLYlink written 4.3 years ago by manekineko130
0
gravatar for David Langenberger
4.3 years ago by
Deutschland
David Langenberger9.1k wrote:

Check this post: miRNA mapping rate is very low.. (less than 0.03%)

ADD COMMENTlink modified 4.3 years ago • written 4.3 years ago by David Langenberger9.1k

thanks for guiding me to that question. Let me try your suggestions first

ADD REPLYlink written 4.3 years ago by prp29150

But please think about your experiment first and do not just follow the suggestions. Probably you have a complete different protocol.... read publications, talk to the wet-lab guys and try to really understand, what they did and read publications! Try an error can be very dangerous in bioinformatics, since you always get results back. If they are good, or f...ed up, you can only decide, when you really know what you are looking at!

ADD REPLYlink written 4.3 years ago by David Langenberger9.1k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1691 users visited in the last hour