Hoping the community can provide some insight. I manage a NGS core facility and recently did some miRNA-Seq for a user. The library kit was NEB Next Small RNA and I sequenced on the MiSeq generating 50 bp reads. Now...the user tells me that 3 of the samples are mir-21 Knock-Out mice confirmed by PCR however I get reads mapping to mir-21a in ALL of the samples. It is significantly down-regulated in the Knock-Outs but there are plenty of reads there nonetheless. Does anyone have any explanations for this, I'm at a loss.
If it helps here is the Bioinformatic pipeline I am using:
-CutAdapt to trim low quality reads and adapters -Bowtie to map to GRCm38 -HTSeq to count reads mapping to miRNAs -DESeq2 for differential expression
Thanks everyone!
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can you please show us a few reads mapping mir-21 ?
Sure, here is s screen shot from IGV.
Link: https://ibb.co/bJavtQ
no, I want the SAM records please, using samtools, to see the MAQs & other attributes.
I'm not sure how to get you a SAM record. I can get you a BAM file. The SAM file is too large for me to parse on my computer.
When I look at the individual reads mapped to mir-21a they have MAPQs of 255 which I see from the documentation that means the mapping quality is not available.
I just found out that Bowtie does not report MAQ values as other aligners do. For Bowtie a MAQ of 255 simply means it is a unique alignment.
Neat trick, thank you! Here is the command I used:
Here is a few lines of the output, how can I get you the whole file?
I added markup to your post for increased readability. You can do this by selecting the text and clicking the 101010 button. When you compose or edit a post that button is in your toolbar, see image below:
Pierre,
Did you get a chance to look at the SAM record?