I am using Oxford Nanopore RNA sequencing (Direct RNA minION) using total RNA from human cell line but majority of my reads are aligned to mitochondrial genome. Secondly, many reads for the genes in interest are either missing or partial reads are available and all these are at 3' end while in most of the cases the 5' end has no or little reads. Thirdly, this is the case with low expressing genes while highly expressed genes are in general ok. Can anyone suggest their experience with such issues and what possibly the causes could be. Thank you very much.
Hi Asif
RNA degradation starts at 5', leading to the detection by NGS of 3' ends while 5' ends disappear...in addition, cell apoptosis often includes polyA RNA degradation and increase of mtRNA representation, resulting the results you get from your ONT sequencing.
just be sure of viability of the cells from which you extract the RNA, and check for cell apoptosis.
fred
Thanks Frederic Lepretre. Yes, that makes sense. However, my RNA had a RIN=10 while 260/280=2.05 and 260/230=2.23. I checked the expression of genes of interest and housekeeping genes and there was good expression. This is the best I could do to check the quality of RNA. Do you think it is still possible to assume that RNA was degraded? It is interesting that the genes that are highly expressed (as determined by qPCR) had good number of RNA sequencing reads as compared to low expressing genes. Could there be a correlation between low expression and sequencing read?
Regarding apoptosis, I am not sure as I got the frozen cells so I am not aware of the background of the cell health. How can I check for the apoptosis?
with a RIN at 10 your samples are in good shape (no degradation at all), largely enough to run NGS in good conditions. but still the issue with high representation of mitochondrial RNA, keeping not enough "space" for your targets. checking for cell apoptosis is as you say in the history of your project.
and you're also right to correlate NGS results to gene expression. assuming this point, at the lab, we now are running 3' RNAseq on NovaSeq machine to replace Agilent arrays quantification.
all the best
fred
- Badges
- Science topic
- Similar topics
- Chemistry
- Sulfites
- Inorganic Chemistry
- Inorganic Chemicals
- Salts