weird noisy peaks on bioanalyzer

[Julien Roux]

Hi everyone,
We are getting some weird results when running RNA-seq libraries on Agilent Bioanalyzer.

The libraries show a peak at the expected length, but the peak itself is very noisy, with a sort of banding pattern:



We're using TruSeq RNA v.1 kit, following the protocol (except size-selection step before PCR enrichment, but we've seen the same thing without size selection). All libraries generated in the same batch do not display these strange peaks, some have a nice and smooth peak at the expected size.

Do you have any idea what could be going wrong?
Thank you very much in advance!

[NextGenSeq]

Do you shear your cDNA?

Unsheared cDNA can show peaks like this.

[ETHANol]

I've seen this before and it was a problem with the Bioanalyzer. What I don't know but the libraries were fine.

[Julien Roux]

NextGenSeq: Yes, the DNA is fragmented before cDNA synthesis, with the Elute, Prime, Fragment Mix (EPF) provided by Illumina. I'm not sure the problem comes from this step since other libraries of the same batch are not affected.

ETHANol: Don't you think such a problem with the Bioanalyzer would be consistently seen across all 12 wells? This is not what we see in our case. I guess we can try to rerun them on the bioanalyzer...

Thanks for your help

[mnkyboy]

Are all of the other libraries higher in concentration? I have seen in lower concentration libraries the curve is less rounded and you see individual peaks like you are seeing.

Based on your dye peaks and the FU this looks to be a low concentration library.

[Julien Roux]

Actually not. Some high concentration libraries also show a noisy peak, while some low concentration libraries look perfectly fine.
Did you sequence the low concentration libraries you're mentioning?