Hi everyone,
this is not the first time I’ve encountered this issue during the preparation of 16S libraries (Illumina protocol: “16S Metagenomic Sequencing Library Preparation”), so I’d like to take the opportunity to ask for your opinion.
Sometimes, once the libraries preparation is completed (which includes amplicon PCR, clean-up 1, index PCR, clean-up 2), I find that most of the samples appear properly cleaned in the initial part of the Bioanalyzer trace—meaning there are no shorter fragments than the target amplicon. However, in some samples, these shorter fragments are still present (and I would like to avoid that), since they are shorter than the amplicons, they appear at the beginning of the trace.
What seems strange to me is that the final libraries show very high DNA concentration on the Qubit, so the two clean-up steps should be working properly in removing these short fragments, because the longer amplicons should bind more efficiently to the magnetic beads, allowing the shorter fragments to be discarded with the supernatant.
Also, I’m not sure whether these fragments (whose nature is unknown to me) could cause issues during sequencing, such as unexpectedly increasing the molarity of the final pool and thus the cluster density.
Do you have any idea why these fragments sometimes remain? Should I change something in the libraries preparation?
I’m attaching images (close-ups of the initial part of the trace) of a clean sample and one showing the issue.
this is not the first time I’ve encountered this issue during the preparation of 16S libraries (Illumina protocol: “16S Metagenomic Sequencing Library Preparation”), so I’d like to take the opportunity to ask for your opinion.
Sometimes, once the libraries preparation is completed (which includes amplicon PCR, clean-up 1, index PCR, clean-up 2), I find that most of the samples appear properly cleaned in the initial part of the Bioanalyzer trace—meaning there are no shorter fragments than the target amplicon. However, in some samples, these shorter fragments are still present (and I would like to avoid that), since they are shorter than the amplicons, they appear at the beginning of the trace.
What seems strange to me is that the final libraries show very high DNA concentration on the Qubit, so the two clean-up steps should be working properly in removing these short fragments, because the longer amplicons should bind more efficiently to the magnetic beads, allowing the shorter fragments to be discarded with the supernatant.
Also, I’m not sure whether these fragments (whose nature is unknown to me) could cause issues during sequencing, such as unexpectedly increasing the molarity of the final pool and thus the cluster density.
Do you have any idea why these fragments sometimes remain? Should I change something in the libraries preparation?
I’m attaching images (close-ups of the initial part of the trace) of a clean sample and one showing the issue.