Question: Peak calling for microRNA
1
gravatar for JulianC
11 months ago by
JulianC10
JulianC10 wrote:

Hi everyone!

I'm working with microRNA, I have miRNA-Seq from different tissue and I want to perform DEA among them, but only considering a small genomic region. I aligned my reads against my reference, and I visualized them on IGV. The peaks I see do not correspond to any annotated miRNA for my reference genome, so I have not an annotation. In order to perform DEA, I want to create a Count Table containing the number of reads for each peak I see in my region of interest (I want to use it for DESeq2) . How can I do this peak-calling operation for mapped miRNAs? I have only the BAM and SAM files derived from the alignment (performed with Bowtie2) and the reference genome (FASTA). Thanks in advance!

mirna • 338 views
ADD COMMENTlink modified 11 months ago by Devon Ryan91k • written 11 months ago by JulianC10

Have you looked at the repeatMasker track for that area on the UCSC genome browser? It's not unheard of to get a bunch of other ncRNAs (fragmented rRNAs, etc.) in smallRNAseq datasets? Are these the appropriate length for miRNA in your species?

ADD REPLYlink written 11 months ago by Devon Ryan91k

In UCSC genome browser there's not the new assembly for my species, not yet. However from NCBI report I know that in my species there are 728 annotated miRNA (but I think I found different ones) with a mean lenght of 23 bp. My problem is essentially to get the read counting for the peaks I see on IGV (few peaks actually)

ADD REPLYlink written 11 months ago by JulianC10
0
gravatar for Devon Ryan
11 months ago by
Devon Ryan91k
Freiburg, Germany
Devon Ryan91k wrote:

There are probably some miRNA specific tools for this, but worst case scenario you could use a peak caller like MACS2. If you use that, you'll need to use something like --keep-dup 1000000 to ensure it keep the big stacks of reads you're likely seeing intact. You might have to filter the results a bit to remove false negative, or possible prefilter your BAM files if you have a bunch of alignments of the wrong length (you might be able to use alignmentSieve from deepTools for this, though I don't think I've ever tested it with single-end reads), but in general I would think that that should work.

ADD COMMENTlink modified 11 months ago • written 11 months ago by Devon Ryan91k
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