Question about library quantification prior sequencing

[boyfwl]

Hi There,

I have recently encountered a problem about library quantification that has hindered my project for months. And i have browsed through all the questions here, it seems this has never happened to anyone before. I really hope if anyone knows how to solve this. Many thanks

After i generated my library, i did:
1: qubit to measure concentration, worked fine, concentration between 80-300 ng/ul
2: did a TA clone and sent 20 clones for sequencing to look at the accuracy of primer sequence, more than 80% are 100% accurate.
3: Did a regular PCR using illumina primer set (p5/p7) under a serial of anneal temperature (54-70), got very strong product at the correct size.

The problem comes when i use KAPA qPCR kit, i used a 4pM and 2pM (adjusted according to qubit result) as template, the result always showed less than 1% of the library was recovered. I should also mention that i included a control sample from someone else when i did all the above experiments. They worked the same in the first three, but differ at qPCR, with my sample came out much much lower than the control.

This has been driving me crazy, please help.

[mhapp95]

How are you preparing the samples? (kit, custom protocol, etc.) I ran into a somewhat similar issue when I ordered my own adapters and accidentally flipped the p7 orientation. It still duplexed readily with the P5 (somehow), and ligated well to my samples. You could see a ~120 bp jump in band size on a gel between my samples before and after ligation. Great qubit and tapestation concentrations as well. Then I went to use the Kapa qPCR kit, and even the undiluted library wasn't above the lowest standard. What you're describing sounds like an issue with proper adapters/proper adapter ligation, as qPCR will only measure what will be able to cluster on the sequencer.

[boyfwl]

Hi mhapp95,

Thanks for your reply.

It is a whole genome GECKO V2 CRISPR knock out screening. The library was generated by two step PCR from whole genome of a bunch of culture cells. DNA was collected for gel extraction after the second step PCR. As i mentioned, i did a TA clone and sequencing to my PCR product, and the result showed majority of it is my aiming product (with correct p5/p7 and correct length).

I indeed sorted of solved the problem yesterday. I did a bunch of regular PCR using p5/p7 with a serial dilution of the templates (1 from my own and 1 valid control). It turns out my sample is 30-100 times less efficient comparing to positive control. Then i went back to qPCR and used 100 times more template and the output was good enough.

I still didn't figure out what the problem was. Judge from what we had, the only viable answer will be incorrect measurement by QUBIT of my sample, which resulted in a 100 times overrate of concentration.

Thanks again.

How are you preparing the samples? (kit, custom protocol, etc.) I ran into a somewhat similar issue when I ordered my own adapters and accidentally flipped the p7 orientation. It still duplexed readily with the P5 (somehow), and ligated well to my samples. You could see a ~120 bp jump in band size on a gel between my samples before and after ligation. Great qubit and tapestation concentrations as well. Then I went to use the Kapa qPCR kit, and even the undiluted library wasn't above the lowest standard. What you're describing sounds like an issue with proper adapters/proper adapter ligation, as qPCR will only measure what will be able to cluster on the sequencer.