Question: How to get the rRNA ratio from a RNAseq dataset
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gravatar for niu2rseq
4.2 years ago by
niu2rseq70
United States
niu2rseq70 wrote:

Hello,

 

I want to know if there is any way using the bedtools and miRdeep2 output bed file to get the rRNA ratio in my miRNAseq fastq data. Thank you very much!

 

I have a gtf file, a genome.fa, a bed file from the miRdeep2. Thanks!

ADD COMMENTlink written 4.2 years ago by niu2rseq70
1

Why don't you just align to an rRNA fasta file and then count the reads? That'd be quick and easy.

ADD REPLYlink written 4.2 years ago by Devon Ryan86k

Thank you for this idea! Here is what I did:

 

I downloaded the sequence  M_ncrna.fa from Ensembl.

And from this I pulled out all rRNA sequences here: 

awk '/rRNA/{print $0;getline;print $0}' M_ncrna.fa > M_rRNA.fa

and then I build a bowtie2 index for this M_rRNA.fa

and then I did: bowtie2 M_rRNA my.fastq -S aligned.sam

I don't really care about the sam file but from the output oe file I got:

1684608 reads; of these:

  1684608 (100.00%) were unpaired; of these:

    1680288 (99.74%) aligned 0 times

    48 (0.00%) aligned exactly 1 time

    4272 (0.25%) aligned >1 times

 

0.26% overall alignment rate

 

Does this make sense? I doubted it but I am not sure where the problem is.  Any suggestiosn?

ADD REPLYlink written 4.2 years ago by niu2rseq70

Without checking, my guess is that your awk command isn't sufficient to print the entire sequence. rRNAs can be quite large, so you'll miss a lot of sequence unless it's all on one line.

ADD REPLYlink written 4.2 years ago by Devon Ryan86k

Hi Devon, 

Here the ncrna.fa is quite small, only 998K. It contains only 7228 lines and 3614 sequences. There are only 504 rRNA sequences there. 

 

ADD REPLYlink written 4.2 years ago by niu2rseq70

Fair enough then. That does seem to be too low an alignment number, though. Are these raw reads or is this after collapsing things?

ADD REPLYlink written 4.2 years ago by Devon Ryan86k
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