BackgroundVibrio 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 ofVibrio spp.at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantificationviadigital PCR (dPCR).ResultsWe developed six DNA standards, calledVibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA librariesviadPCR. To enableVibrio-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 quantifyVibrio-DNA in a pooled DNA mixture derived from severalVibriospecies in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR.ConclusionsWe 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.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09429-8.