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seqtechno1 05-16-2019 07:42 AM

high percentage of reads mapping to tRNA?
How do you interpret this? What does it mean for wet-lab side of things, does it inform any step that went right or wrong? This is for a total RNAseq minus rRNA experiment. :confused::confused::confused:

UCan'tBcereus 05-29-2019 10:56 AM

Hi seqtechno1,

Issues could be:

-Ribodepletion Kit used does not target tRNAs. If they are targeted then they will not be removed. Most kits use a pretty high (2.0x or above) bead cleanup, so even very small tRNAs would make it through the ribodepletion process. You could reduce the bead ratio so that tRNAs are removed but mRNA stays (assuming your mRNA is high enough quality to not be mixed in with the tRNAs.

-If this is the case, and most of your rRNA deleted RNA is tRNA, then when you go to do cDNA synthesis, most of your cDNA will be from tRNA, not your desired mRNA. Most RNASeq library prep kits will have various bead cleanups at various ratios. If you want, you try reducing the bead cleanup ratios at these steps as well to try and remove as much small cDNA products synthesized from small tRNA as possible.

These ideas will only work if your mRNA is go high enough quality to not be mixed in with tRNAs. IE if you are working with FFPE Total RNA, then its a lot harder to troubleshoot.

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