Discripancy in mirdeep2.pl and quantifier.pl outputs.

[oneito]

I am working with miRNA from a non model organism. I want identify both known and novel miRNAs. I have five replicates for each condition. Because my organism has no annotated miRNA, I used its close relative to identify known and novel miRNAs with the code

miRDeep2.pl A_collapsed.fa A.fasta A_collapsed_vs_genome.arf miRBase_dme_v14.fa none none 2>A_report.log

This gives total and unique reads that map to each identified mature miRNA and star sequences. But now when I use the results from mirdeep2.pl output to feed into quantifier.pl for expression analysis, I find that some of the most abundant miRNA (high number of reads mapped according to mirdeep2.pl) are not expressed (zero counts). The code for quantifier:

quantifier.pl -p ./for_qiantifier/precursor.fasta -m ./for_qiantifier/Mature.fasta
-r ./GffI_cat-reads_collapsed.fa
-s ./for_qiantifier/Star.fasta -y now

I am wondering if this is normal or there is something wrong somewhere. Some insight please.

[mschilli87]

Without being able to reproduce your results (by shared code and data) it will be very hard to provide any help tracking down your problem. For example, how did you generate the precursor.fasta and Mature.fasta? If they don't contain that abundant miRNA you mentioned, quantifier.pl won't find assign reads to it. Also, you mention replicates and conditions but don't explain how you combine them. I'd pool all data for the annotation step and run the quantification for each sample separately. Also, how did you 'use its close relative'? Finally, consider looking for help at Biostars, Stackexchange or similar sites because this is a bug tracker. As longs as you don't have some strong suspicion that there is something wrong with miRDeep2 that requires fixing, this is likely the wrong place to as for help.

[Drmirdeep]

Please provide log file/terminal output from the quantifier call