Genomic DNA integrity importance in amplicon sequencing

[Ayaka]

Hi there, in my lab we are going to be sequencing 16S with Miseq from stool samples and we are currently in the DNA extraction step.

We are using Qiagen's QiAmp DNA stool mini kit and have performed an additional step of bead beating that gives us twice of the final DNA concentration. The issue is that when we check the integrity we see smear bands.

How important is if we want to see diversity to start with integral DNA?

We think is important, since we don´t have a way to control what DNA is being degraded and we might loose 16S of some bacterias, but we have performed RFLP-PCR and the profiles are very similar and wanted to ask for your experience.

Thanks!

[Baseless]

When sampling DNA from stool I'd expect a stromg degree of degradation and thus a high spread of sizes on gel/bioanalyzer. Even if you have DNA from blood and purify, you would rather see a smear in a certain range typical for your purification method than a sharp peak we know from (most) PCR products. What I would question is, if you do your PCR for whatever 16s fragment you intend to sequence - do you get a PCR product of an appropriate size range?

[Ayaka]

Hi Baseless thank you for answering. We indeed get a PCR product of the appropriate size. A person from a sequencing center told me that as long as the smeared bands are above the size of the PCR product you want to amplify it would be Ok.
I still think it could cause loss of diversity though, because of the random fragmentation of the DNA, although I guess it might not be such a big deal...

[Baseless]

And one more thing to consider on the final product: the 16s amplicons are a diverse thing, so they are allowed to differ a bit in size.
I would not tolerate this from a PCR amplicon for resequencing of a defined region like for deep-coverage SNV validation, but a degree of size range is ok with me on 16s, TCR and BCR variable regions.
However, never let your guard down on small fragments on adapter dimers, they only lead into trouble.