no alignment from plasma derived small RNA

Question

[memento1984_1]

Hi all, I have this strange alignment issue - source input, amount and quality of RNA seemed to be allright before the libraries were made and libraries also produced enough, good quality library (visible by size, not adapter only library). After sequencing (no issue here) I removed 3' adapter reads using trimmomatic (ILLUMINACLIP:./mirna.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:15) library prep used is QIAprep miRNA.

Tried bwa mem grch37 1.fastq.gz > 1.sam

Just upon inspection of sam file there are no aligned reads. Any ideas?

[GenoMax]

You seem to have used mirna.fa during trimming, which presumably has the kit specific adapter you had for your libraries. So trimming of the reads is not the issue here (are the reads in the range you would expect for small RNA)? You may want to take a few reads and do a quick blast to make sure they align to the correct species.

You want to do ungapped alignments with small RNA, so trying bowtie v.1.x may be an alternate option to try.

[memento1984_1]

yes, I checked the distribution of the fragments and it is what would be normally expected. I tried bowtie2 and the alignment is there: ~9% reads with only 1 mapping in the human genome ~39% reads with >1 mapping ~52% no alignment

Is this normal? What would be the best way to get RPKM like count for each mir??? Thank You!