Optimization of the cDNA-PCR sequencing protocol​

[BobArchuleta]

I'm using the cDNA-PCR Sequencing Kit (SQK-PCS111) to sequence full-length transcripts from human brain tissue. I have followed the protocol from Oxford Nanopore Technologies and I have obtained about 10 ng of cDNA per sample. However, when I run the QC step using the Qubit and the TapeStation, I see a very low concentration of cDNA (around 1 ng/ul) and a very broad size distribution (from 200 bp to over 10 kb). How can I optimize the cDNA-PCR sequencing protocol to get higher yield and quality of cDNA?

[gringer]

Most optimisation around PCR sequencing is around optimising the PCR itself.

If your yield is about 1/10th of what you want, then as a simple initial fix try running the PCR for four more cycles.

A broad size distribution is typical for cDNA sequencing; we usually get a similar range (i.e. 0.2kb - 10kb), averaging 0.6 - 1.5 kb per read depending on how good the input RNA is. Fresh is best - better, less degraded RNA will produce longer reads, more reads, and a more productive sequencing run overall.