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!
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!
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