Abstract
BackgroundOne 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.ResultsUsing 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 greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts.ConclusionOur improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.
Highlights
One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step
The very same samples were processed after the 2nd-step PCR by a further digital droplet PCR (ddPCR) step
By comparing the resulting sequences of both procedures, we evaluated whether the ddPCR step introduces bias
Summary
One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. For low-biomass samples, insufficient or even commonly unde‐ tectable DNA amounts can limit or prohibit further analysis in standard protocols. In 1985, the 16S rRNA gene was described for the first time as a molecular tool for identifying microbes that were previously shown to be unculturable [1]. This ubiquitous bacterial gene possesses special features containing conserved regions that enable primer binding and amplification, as well as hypervariable regions allowing phylogenetic differentiation. Easy to lyse Gram-negative bacteria are favored by several extraction methods compared
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