I am working with miRNA-seq dataset from a non model organism with no known miRNA. As such I have identify both known and novel miRNAs and I am using miRDeep2. I have five replicates for each condition. Because my organism has no annotated miRNA, I used mature miRNA from a close relative (D. melanogaster) to my organism to identify known and novel miRNAs. To do this, I combined all the sequencing data into one and used the code below for identification of miRNAs.
miRDeep2.pl A_collapsed.fa A.fasta A_collapsed_vs_genome.arf none miRBase_dme_v14.fa none 2>A_report.log
This identified known and novel miRNAs with their associated Total and unique number of reads that mapped to them. Sorted the miRNA based on the miRDeep2 score >2, randfold p-value = Yes, and at least 10 reads mapping. This now acts as my reference.
How when I used the mature miRNA, Star consensus, and precursor sequences from the miRDeep2 output (my reference) to run quantifier.pl for miRNA expression for each replicates, I run into a problem where some of the miRNAs with thousands of reads (over 1000 counts) mapping to them have zero reads (zero counts) with quantifier.pl. The code for quantifier.pl:
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.
Again, when I run initial code for miRDeep2.pl for each replicate, I realize that some mature miRNA are identified in different parts of the genome different from the one identified when combined seq data is used. How do I reconcile this.
Finally in my output results for both miRDeep2 and quantifier.pl, the .cvs files do not have input IDs or species IDs if I just put some. I am wondering if this should be the case now that the organisms I am working with has no known miRNAs.
Finally, final, I also get something that looks like ‘error’ but not when I run quantifier.pl:
Can't use string ("45") as a HASH ref while "strict refs" in use at /home/ea432/.conda/envs/umi_mir/bin/make_html2.pl line 658.
Can't use string ("57") as a HASH ref while "strict refs" in use at /home/ea432/.conda/envs/umi_mir/bin/make_html2.pl line 658. Etc.
Some help please. Thanks.
Sorry bingyanzheng18 I was not able to solve it. but when I eliminated -s Star.fasta it improved the mapping counts. You can email the authors of mirdeep2 for help. They should be able to help.