miRNAseq analysis not shown adapter sequence and huge N’s content
This is my third time doing miRNA sequencing analysis, so i do not have huge experience on this…
So, i have 18 human semen samples, (also no experience in this type samples) i have been reading alot of papers about mirna and human semen samples and its difficult to have and to know if the samples have a great quantity and quality of RNA to perform the analysis. However, my colleagues with permission of the supervisor went forward and sequenced using the NEXTFLEX Small RNA-Seq Kit v3.
My struggling is right on raw data, with bad quality that i realized was because of huge percentage of N’s content (~10-22% in the samples) i mean i have reads that is totally N! Never seen this!
Also, to get more fun, adapter sequence from the kit is not detected in any sample. I tested the mirtrace software to test the quality of the samples, the software uses several adapters sequences (included the Nextflex) and its not detected any adapter. However, mirtrace detects some miRNAs on samples, so i run that Quickmirseq to perform differential expression, i got 65 mirna in matrix of counts!
Moreover, i simple aligned with bowtie the samples with human reference hg38, that 90% of reads were unmapped 🙁 i blast the unmapped and gives match with several organisms. I am stuck right now!
I wonder if anyone came with is similar situation, what can i do more, probably the rna extraction not went well right? Any thoughts?
Thanks in advance.
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