QC of cDNA library prep for GA

[cybog337]

we have tried RNA seq 4 times and usually we faced low cluster problem. we tried to solve that with various approaches such as qRT-PCR using phix or internal controls, and bioanalyzer. However, this methods didn't wokr. some sample was working but most samples were fewer clusters. please, give any word to me if you had same troubles.

Thank you!!

[OnUrMark]

Hi Cybog337,

I know this is an older thread, but I am having a similar problem. Are you using a do-it-yourself method?

Thanks

[NextGenSeq]

We get good results using RiboZero and the Epicentre ScriptSeq kit. It's worked everytime for us.

[OnUrMark]

samples amplify during PCR Enrich but don't sequence

Hi NGS,

I am using a DIY method, and we would really like to solve the problem so that we can use the data collected thus far. If I change methods, there will be a different bias, and this will give false positives for our gene expression study.

I have some interesting clues as to what is wrong. I get great amplification during PCR enrichment. I gel extract, confirm quality on bioanalyzer, and then I use the Qubit to determine each sample's concentration. I mix equal molar concentrations of 6 barcoded samples together and submit for GA sequencing.

The results, highly variable cluster densities among multiplexed samples and between lanes of different samples. It does not appear that the barcodes being too similar is the problem. I have checked some samples using qPCR so the individual barcodes should not effect the results. The samples with a high number of reads gave much higher values than those with low reads. So there are a lot of ~350bp fragments in my libraries that are amplifying during PCR enrichment but are not sequencing. I believe they are binding to the flow cell (but not certain) because increasing the sample concentration is not solving the problem. I have tried another lab's adapters and primers with only slightly better results.

I am about to do a pcr enrichment to check before and after fragment lengths. I have very limited sample, and I'm not sure if pre-pcr enriched sample will actually show on the bioanalyzer.

Does anyone have an idea why a sample would amplify during pcr enrichment but not sequence?

Thanks!

Attachment1: bioanalyzer of pcr enriched and gel extracted sample that did not sequence
Attachment2: gel of post-pcr enrichment/pre-gel extraction samples, some sequenced and some did not[ATTACH][/ATTACH]