Tap water qualification is quite important for human health, the DNA extraction from tap water is a current challenge since the microbes inside is limited. This article aims to evaluate various DNA extraction methods and based on the different reagents or procedures and their influence to do some modifications for better extraction. DNA yield, purity and fragment size are three evaluation criteria for extracted DNA, while the reproducibility and operational convenience are also taken into evaluation for each method. According to the results, extraction methods could be recommended for different situations. It is concluded that commercial kits show better reproducibility in yield and quality aspects and more convenient in operation than phenol/chloroform based methods. The modified method which adds enzymatic and chemical lysis method into standard commercial kit protocol, shows the highest DNA yield. The phenol/chloroform based methods give the best average purity, especially for protein contaminants. Considering the fragment size, the DNA extracted from Quick-DNA HMW MagBead kit (ZYMO Research, USA) distributes widest with more HMW DNA among all methods. The results presented here suggest that that the DNA extraction method of choice for tap water samples should be the modified Quick-DNA HMW MagBead kit.

Drinking water safety is critical to public health and the stability of drinking water quality is highly related with drinking water distribution systems (DWDSs). Antibiotic resistance genes (ARGs) and their hosts are the two major concerns in drinking water, however, the knowledge of fate of ARGs and their hosts were limited due to the current short-read length based sequencing technologies. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) is expected to overcome the challenges on ARGs and ARB characterization owing to its ultra-long reads generated. In this study, therefore, direct gDNA Nanopore sequencing was performed on treated water, distributed water and biofilm samples in two different DWDSs located at Kamerik and Lekkerkerk to explore its potential on ARGs and ARB characterization in drinking water systems. Additionally, the performances of taxonomy classification and ARGs profiling with Oxford Nanopore gDNA sequencing with different thresholds were assessed using an artificial microbial community. However, the conflict between the high requirements of input DNA quality and quantity with Nanopore sequencing and extremely low microbial biomass content in drinking water samples led to a challenge for our research. Therefore, evaluation on different sample preparation methods was conducted to meet the requirements of the input DNA.