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miR sequencing: miR and miR*, sense and antisense das RNA Sequencing 0 10-21-2009 06:27 AM

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Old 06-15-2017, 07:36 AM   #1
gkuffel
Genomics Lab Mgr.
 
Location: Chicago

Join Date: Oct 2012
Posts: 14
Default Read mapping to mir-21a in a mir-21 Knock-out mouse

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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Old 06-15-2017, 01:49 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,226
Default

I think the issue is biological. Majority of techniques referred as knock out are actually knock down so there is still some transcrptional activity from target genes.

Most of knock out transgenes cannot be confirmed by a simple PCR. They should use qPCR with the same sample usd for ibrary prep for knock out confirmation. There is also possibility of cross contamination during library or RNA sample prep.
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