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Old 11-07-2017, 02:15 AM   #1
frymor
Senior Member
 
Location: Germany

Join Date: May 2010
Posts: 149
Default library depths for sequencing

Hi everybody,

I was approached by a biologist in our lab and was ask a specific question. As I was not really sure, I would like to share it with the community and hope to get some answers from more experienced users.


They would like to run an RNA-Seq experiment to find out more about replication processes in yeast. The difficulty they're having is this.
During the replication process, the DNA is duplicated in the specific positions, where the replication is currently taking place. But this is not at all cells at one specific time nor place nor speed ( AFAIK, correct me if I am wrong please), as it has multiple origin of replications.

THe question was how deep should a library be, so that these changes can be identified.
If for the sake of argument we have a replication process in 10% of the cells in the sample, so we get an increase of DNA from 100% to 110%. Is it possible to distinguish this kind of changes in an RNA-Seq experiment?

In my opinion, a regular RNA-Seq experiment in yeast for differential expression analysis should have at least 10M reads to identify changes of at least 2 fold-changes.
But in this case I think a much higher coverage is needed to be able to cover the changes in only 10% of the cells in the sample. Is it possible at all to pin-point this kind of changes?

Should a different technology be used for this kind of analysis?

I would appreciate any kind of help, ideas or references to published experiments.

Thanks in advnace

Assa

P.S.
If this is not the right forum for this kind pf question, please refer me to a better source.
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