Tweet
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. 
-
-
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.
-
The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...Yesterday, 07:48 AM -
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...04-22-2024, 07:01 AM
Comment