Mapping Ribo-Seq and mRNA-Seq from the same project

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Assa Yeroslaviz

Apr 1, 2022, 7:25:53 AM4/1/22
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I have a project with both RNA-Seq and Ribo-Seq. I would like to know if it makes more sense to map them with similar parameters or should I use short-reads specific parameters for the Ribo-Seq data?
After trimming and preprocessing the Ribo-Seq data I have reads in varying lengths between 12-63, while the RNA-Seq is all 75nt long. 
In the genome indexing step:
Do I need to create two different indexed genomes for the two data sets?
What would you use as a value for sjdbOverhang = ?

In the mapping step:
Does it make sense to include in the Ribo-Seq mapping also such parameters as:
--winAnchorMultimapNmax 200
--seedSearchStartLmax  15
--outFilterMultimapNmax  8
outFilterMismatchNmax 4 

thanks in advance


Alexander Dobin

May 2, 2022, 3:02:28 PM5/2/22
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Hi Assa,

sorry for the delayed reply.
with a highly variable read length for ribo-seq, it's hard to select an ideal value for sjdbOverhang.
I think simply using 74 for both RNA-seq and Ribo-seq will work fine.

Same is true of other parameters. If you are serious about optimizing the parameters, I would recommend selecting a metric of merit (e.g. % of mapped reads) and varying the parameters to optimize it.
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