Question: While generating the % of mapped reads to all miRNAs, do I need to consider unique or multi-match reads?
gravatar for bioinforesearchquestions
2.9 years ago by
United States
bioinforesearchquestions280 wrote:

Hi folks,

I am trying to create a table for the distribution of mapped RNA sequenced reads across miRBase v.21 for each isomiRs.

Commercial Photography

I am using contents from this miRBase.mrd file to generate the reads mapped to isomiRs. I have copied the sample output from miRBase.mrd file generated using miRDeep2.

Commercial Photography

I have written a perl code to identify the isomirs using miRBase.mrd file.

Commercial Photography

As per the above output, some of the sequenced reads are present in both "ssc-let-7a-1" and "ssc-let-7c".

For example,




Note: seq_3228221 is ID and x43 refers to 43 times this sequence present in the FASTQ file.

I am aware that miRDeep2's mapper file uses following bowtie command "bowtie -p 1 -f -n 0 -e 80 -l 18 -a -m 5 --best --strata " to generate the alignment file. This command allows the same sequence to map to multiple miRNAs.

While generating the percentage of mapped reads for all miRNAs, do I need to consider only unique reads (seq_2697760_x253 only once) or reads with multiple matches then it becomes 2*253=506 reads?

For reference, this is the miRDeep2's final read count output file for all miRNAs

Commercial Photography

mirna mirdeep2 rnaseq • 898 views
ADD COMMENTlink modified 2.9 years ago • written 2.9 years ago by bioinforesearchquestions280

I think you should count every read only once. It may map to 100 miRNA, but it is still one read. (I understand you are talking about "sequences" and not reads. I assume that "sequences" are just clustered identical reads, thus the situation doesn't change). The best thing, however, would be to do a very small experiment, if you have time. Try to do the analysis with only 1 sequence (mapping in multiple positions). From there, you will easily understand what is the correct way of treating it.

ADD REPLYlink written 2.9 years ago by Fabio Marroni2.7k

You will need a higher coverage to validate isomiRs, really higher.

ADD REPLYlink written 2.9 years ago by Buffo1.8k
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1924 users visited in the last hour