DdPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples.

ABSTRACT:

Background

One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.

Results

Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a gre

SUBMITTER: Abellan-Schneyder I 

PROVIDER: S-EPMC8684222 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature