Coverage required for 3' end RNA-seq methods

Question

[Aspire]

Methods such as CEL-Seq/CEL-Seq2 sequence only the 3' end of a transcript. I was told that the sequencing depth required for such methods is less than the sequencing required for standard RNA-Seq (which counts fragments from the whole transcript).

Why is this so (in case you confirm)?

Answer 1

[benformatics]

If you have a mRNA transcript that is 20kb in length but your protocol only involves sequencing 2kb from the 3' end then you are essentially using only 10% of the sequencing compared to a protocol that sequences the full-length transcript.

The number of molecules doesn't change so the cost of sequencing would be the same for:

• 1 full-length transcript of 20kb
• 10 three-prime-UTR transcripts of 2kb each

[benformatics]

In regards to coverage calculations you are shrinking the number in the denominator (size of reference sequence) since you no longer get any reads from the 5' end of long genes.

[Carlo Yague]

This is a good point. Another thing to consider, is that by doing CEL-seq or any poly(A) based assay, you select against non polyAdenylated RNA (including rRNA and many other abundant ncRNAs), so the relative proportion of reads mapped against protein coding genes can be higher than with total RNA-seq.