Hello,
I am interested in using RNA-seq data to identify fusion genes. To do this you need a certain number of reads supporting a given fusion, maybe 10 or so. I am trying to figure out in a systematic way what RIN number is a good lower cutoff for notifying my sequencing provider that they should switch to a ribosomal depletion kit over a poly-A pull down kit. I know about the 3' bias that you get with poly-A pull down if you have degraded RNA. Looking at some coverage data, it looks like there could be a general relation between an exponential decay function and the transcript length that is a function of the RIN number. Has anyone tried to quantify something like this? The problem with just jumping ship over to a ribosomal depletion kit is that I have seen fairly low mapping rates to coding genes in those kits, on the order of 10-20% of all reads. The rest of the reads land in introns or other places on the genome. So say you drop coverage from 1000X down to 100X 3' to 5' in an average length transcript. Isn't that better than a kit where you can only hope for 100X (10X lower) across the entire transcript? So that question is what lead me to want to have this quantified somehow as a function of RIN, so I can make a better decision about when to switch between the two preps based on my needs.
Is anyone aware of such a study?
Thanks,
John
I am interested in using RNA-seq data to identify fusion genes. To do this you need a certain number of reads supporting a given fusion, maybe 10 or so. I am trying to figure out in a systematic way what RIN number is a good lower cutoff for notifying my sequencing provider that they should switch to a ribosomal depletion kit over a poly-A pull down kit. I know about the 3' bias that you get with poly-A pull down if you have degraded RNA. Looking at some coverage data, it looks like there could be a general relation between an exponential decay function and the transcript length that is a function of the RIN number. Has anyone tried to quantify something like this? The problem with just jumping ship over to a ribosomal depletion kit is that I have seen fairly low mapping rates to coding genes in those kits, on the order of 10-20% of all reads. The rest of the reads land in introns or other places on the genome. So say you drop coverage from 1000X down to 100X 3' to 5' in an average length transcript. Isn't that better than a kit where you can only hope for 100X (10X lower) across the entire transcript? So that question is what lead me to want to have this quantified somehow as a function of RIN, so I can make a better decision about when to switch between the two preps based on my needs.
Is anyone aware of such a study?
Thanks,
John