Double peaks on Bioanalyzer? 16s V3-V4 amplicon

[vangFO]

Hi Everyone,

I am looking for a theroretical and practicalexplanation here because I´m new to NGS. We have been using the standard Illumina 16s metagenomics protocol that amplifes a 540 bp fragment of the V3-V4 hypervariable region. Here are my questions.

1. Sometimes we have sharp bands larger than our expected amplicon. Is this primers annealing to repeat areas of the DNA? So it isn´t an exact match but enough to bind at high cycle number? or is this imcomplete ampification that is hanging onto something else?

2. I see alot of other people posting about double peaks...sometimes we see them very close together at the expected amplicon size. Is this a structural artifact? like supercoiling of the amplicon?

3. For some samples the Qubit reading was 3-4x higher than the bioanalyzer.
even for samples with clean bands. What could be the reason for this?

I have attached a PDF with examples of our bioanalyzer readout.

Thank you so much for your help!

[itstrieu]

How many cycles did you use for the PCR reaction? It might be that your primers are exhaust and your PCR products are annealing to each other. See this link: https://dnatech.genomecenter.ucdavis...e-pcr-bubbles/

For the first PCR reaction, we use 21 cycles and for the second PCR reaction we use 8. Attached are typical traces we get for the amplicon and final library. As for the concentration that the bioanalyzer gives, I would trust the concentration that the Qubit is giving since it will be more accurate.