Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp

Abstract

Background Vibrio spp. are a diverse group of ecologically important marine bacteria responsible for several foodborne outbreaks of gastroenteritis around the world. Their detection and characterization are moving away from conventional culture-based methods towards next generation sequencing (NGS)-based approaches. However, genomic methods are relative in nature and suffer from technical biases arising from library preparation and sequencing. Here, we introduce a quantitative NGS-based method that enables the quantitation of Vibrio spp. at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantification via digital PCR (dPCR). Results We developed six DNA standards, called Vibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA libraries via dPCR. To enable Vibrio-Sequin quantification, we validated three duplex dPCR methods to quantify the six targets. LOQs were ranging from 20 to 120 cp/µl for the six standards, whereas the limit of detection (LOD) was ~ 10 cp/µl for all six assays. Subsequently, a quantitative genomics approach was applied to quantify Vibrio-DNA in a pooled DNA mixture derived from several Vibrio species in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR. Conclusions We significantly advance existing quantitative (meta)genomic methods by ensuring metrological traceability of NGS-based DNA quantification. Our method represents a useful tool for future metagenomic studies aiming at quantifying microbial DNA in an absolute manner. The inclusion of dPCR into sequencing-based methods supports the development of statistical approaches for the estimation of measurement uncertainties (MU) for NGS, which is still in its infancy.

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38

“…FNV38. More than 100 Vibrio species have been identified to date and they are ubiquitous gram-negative bacteria, found in a wide range of marine and aquatic temperate habitats (Flütsch et al 2023). Vibrio FNV38 was identified as part of a screening activity undertaken to mine organisms associated with Ulva thalli for ulvan and cellulose saccharifying enzymes (Rodrigues et al 2017) where it was shown to have activity on these substrates.…”