Question: While generating the % of mapped reads to all miRNAs, do I need to consider unique or multi-match reads?
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16 months ago by
United States
bioinforesearchquestions250 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.

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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.

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

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

For example,

seq_3228221_x43

seq_3218517_x44

seq_2697760_x253

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

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mirna mirdeep2 rnaseq • 483 views
ADD COMMENTlink modified 16 months ago • written 16 months ago by bioinforesearchquestions250

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 16 months ago by Fabio Marroni2.3k

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

ADD REPLYlink written 16 months ago by Buffo1.6k
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