Swiss Federal Institute of Metrology METAS, Lindenweg 50, Bern-Wabern, 3003, Switzerland.
Swiss Federal Institute of Metrology METAS, Campus Liebefeld, Schwarzenburgstrasse 165, Bern-Köniz, 3097, Switzerland.
BMC Genomics. 2023 Jul 4;24(1):375. doi: 10.1186/s12864-023-09429-8.
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).
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.
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.
弧菌属是一组生态重要的海洋细菌，它们是导致世界范围内几起食源性肠胃炎暴发的罪魁祸首。它们的检测和特征分析正在从传统的基于培养的方法转向基于下一代测序（NGS）的方法。然而，基因组方法具有一定的局限性，并且受到文库制备和测序产生的技术偏差的影响。在这里，我们引入了一种基于定量 NGS 的方法，该方法通过人工 DNA 标准可实现定量极限（LOQ）下的弧菌属定量，并通过数字 PCR（dPCR）实现绝对定量。
我们开发了六种 DNA 标准品，称为 Vibrio-Sequins，并为它们各自的 dPCR 定量建立了优化的 TaqMan 检测方法。为了实现 Vibrio-Sequin 的定量，我们验证了三种用于在单独测序的 DNA 文库中定量六个靶标的双 dPCR 方法。六个标准品的 LOQs 范围为 20 至 120 cp/µl，而所有六个检测方法的 LOD 约为 10 cp/µl。随后，在一项概念验证研究中，我们将一种定量基因组学方法应用于从几种弧菌属的混合 DNA 混合物中定量 Vibrio-DNA，通过将 NGS 和 dPCR 结合，证明了我们定量基因组学管道的强大功能。
我们通过确保 NGS 基于 DNA 定量的计量溯源性，显著推进了现有的定量（宏）基因组方法。我们的方法代表了一种用于未来绝对定量微生物 DNA 的宏基因组研究的有用工具。将 dPCR 纳入测序方法支持了 NGS 中测量不确定度（MU）估计的统计方法的发展，而这仍处于起步阶段。
