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Old 02-04-2015, 02:20 PM   #3
RNA
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Location: California

Join Date: Aug 2010
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"20X coverage" for RNA-Seq is difficult to define since the copy number varies for transcripts across at least 4 orders of magnitude within a tissue. Therefore estimating "coverage" for RNA-Seq is not nearly as straightforward as it is for DNA applications.

For very highly expressed transcripts, as little as 1 Million reads will easily give you 20X coverage.

But for rare transcripts, you can collect 1 Billion or more reads and still not ever get to 20X coverage.

And of course this issue varies depending upon which tissue you are studying as well...a transcript may be easy to study in liver, but be virtually absent in brain.

For mRNA sequencing (TruSeq Stranded mRNA Kits) we usually recommend 50 Million paired-end 2 X 75 bp reads...you can always go to 100M if you want deeper coverage...but beyond that the cost-benefit ratio of collecting more reads on a single sample really falls off dramatically.
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