Neoprep RNA-Seq troubleshooting

[brcseq]

Hello,

We have had library prep problems using the neoprep for the last 6 runs in a row. It seems like the neoprep library output is failing and/or generating heavily 3' biased libraries with low diversity despite loading 100ng (spec range is 25-100ng) of RNA with RINs of 10, control UHR samples, and samples that previously worked and undergoing 15cycles of PCR.

We have re-bioanalyzed samples after these failures and they always checked out. We changed all our plastic ware and changed lots of reagents.

We have run over 40 neoprep runs without very many problems with the exception of last fall when we had a grouping of similar failed runs. So we thought that perhaps the change in temperature was causing dry indoor air and static. So we humidified the air and grounded all our consumables to dissipate static. Still no luck.

So in general library chemistry, if starting with good input and quality RNA does anyone have any idea how we can be losing so much diversity and gaining strong 3'bias?

[HESmith]

Not specific to the Neoprep but, as a rule, 3' bias in cDNA libraries usually indicates a problem with the RT reaction (or degradation, but it sounds like you've largely eliminated that possibility). I'd contact Illumina tech support to see if other users have reported similar problems from the same lot. I've heard that the Neoprep RNA kits are on backorder, which might also reflect an internal issue with quality (it wouldn't be the first time).

[brcseq]

Hmmm, that is an interesting thought. These 6 runs were actually done with 4 different lots of reagents, but I'm not sure if any of the RT specific reagent lots could have been used across multiple neoprep plate lots. Or perhaps there is a some fluidic issue that isn't properly delivering RT reagents.

Anyway, great idea to try and look into. Unfortunately, it's not always easy to test individual reagents on the neoprep.