One step forwards for the routine use of high-throughput DNA sequencing in environmental monitoring. An efficient and standardizable method to maximize the detection of environmental bacteria

Abstract

We propose an innovative, repeatable, and reliable experimental workflow to concentrate and detect environmental bacteria in drinking water using molecular techniques. We first concentrated bacteria in water samples using tangential flow filtration and then we evaluated two methods of environmental DNA extraction. We performed tests on both artificially contaminated water samples and real drinking water samples. The efficiency of the experimental workflow was measured through qPCR. The successful applicability of the high-throughput DNA sequencing (HTS) approach was demonstrated on drinking water samples. Our results demonstrate the feasibility of our approach in high-throughput-based studies, and we suggest incorporating it in monitoring strategies to have a better representation of the microbial community. In the recent years, HTS techniques have become key tools in the study of microbial communities. To make the leap from academic laboratories to the routine monitoring (e.g., water treatment plants laboratories), we here propose an experimental workflow suitable for the introduction of HTS as a standard method for detecting environmental bacteria.

Keywords: qPCR; DNA extraction; complex matrix; environmental bacteria; high-throughput sequencing; next-generation sequencing; tangential flow concentration; water.

Figure 1

Experimental workflow to test and validate the efficacy of TFF. In the Exp ‐ 1 box, reproducibility was tested (see Section 2.4). The panels Exp ‐ 2A and Exp ‐ 2B report the artificial contamination experiment comparing two different types of DNA extraction (A and B, see Section 2.5). The Exp ‐ 3 panel illustrates the experiment using drinking water samples, instead of artificially contaminated sterile water (see Section 2.5). Finally, the Exp ‐ 4 panel represents the experiment in real conditions using drinking water samples, without artificial contamination (see Section 2.6). Gray cylinders represent the contamination solutions, blue cylinders represent “pre” concentration samples, and dark blue triangles represent “post” concentration samples. Bacteria used for each experiment are listed in white boxes: in green live bacteria, in red dead bacteria, in bold gram‐positive bacteria, and normal font refers to gram‐negative bacteria

Figure 2

Results obtained from the experiments illustrated in Figure 1. On the y axis, the log₂ (DNA copies) is reported. On the x axis, measures obtained from samples before filtration (PRE) and after filtration (POST) are reported. Bacteria used to contaminate water samples are listed in white boxes: in green live bacteria, in red dead bacteria, in bold gram‐positive bacteria, and normal font refers to gram‐negative bacteria. In the case of the experiment with drinking water (Exp ‐ 4) only data after‐filtration are reported