16SrDNA library QC

[Ben_Niu]

Hi everyone,
After the QC by qPCR with my 16SrDNA library, I got a concentration value 13.65nM. When I submitted the sample, it was measured as more than 40nM. The sample is planning to be sequenced by illumina. I don't know if the concentration variation is normal? Do you think the sample is good for illumina sequencing? I look forward to your suggestions.Thank you very much!

[Genohub]

Did you qc more than one sample? How did the other samples compare relative to each other? I've seen these differences before. Did both groups use the same qPCR standards, qPCR mix?

- Genohub

[Ben_Niu]

No, I only did one sample. The experiment was done by a facility, so I've no idea about the qPCR standards or mix. Did you use the samples with such differences for illumina sequencing? Did they work? Thank you!

[Genohub]

Does the facility have a lot of experience with loading samples for sequencing? If yes, then you're probably okay. qPCR quantifies only amplifiable, competent adapter ligated library products. It's used to check cluster density and reduce variability between multiple libraries. My guess is that the qPCR standards or mix used was different. Does your prep have a lot of adapter dimers? If the facility has a lot of experience with NGS, they most likely have experience with cluster density and have optimized their qPCR prep to give them these numbers.

- Genohub

[Ben_Niu]

Thank you so much for the explanation! To avoid the misunderstanding, I want to clarify that 13.65nM is a value obtained with qPCR during QC. I got 40nM with a florescence probes kit before QC. Additionally, before qPCR the result from Tape Station looks good.

[Genohub]

It's fairly common to see discrepancies between double strand specific fluorescent probes and qPCR. dsProbes will bind to anything that has double strands, while qPCR will be more picky and only measure products that can amplify. I haven't seen your Tape Station results but if you're getting a good size range, then it sounds like you're in good shape to sequence.

- Genohub