A comparative assessment of conventional and molecular methods, including MinION nanopore sequencing, for surveying water quality

Abstract

Nucleic acid based techniques, such as quantitative PCR (qPCR) and next generation sequencing (NGS), provide new insights into microbial water quality, but considerable uncertainty remains around their correct interpretation. We demonstrate, for different water sources in informal settlements in the Kathmandu Valley, Nepal, significant Spearman rank correlations between conventional and molecular microbiology methods that indicate faecal contamination. At family and genera level, 16S rRNA amplicon sequencing results obtained with the low-cost, portable next generation sequencer MinION from Oxford Nanopore Technologies had significant Spearman rank correlations with Illumina MiSeq sequencing results. However, method validation by amplicon sequencing of a MOCK microbial community revealed the need to ascertain MinION sequencing results for putative pathogens at species level with complementary qPCR assays. Vibrio cholerae hazards were poorly associated with plate count faecal coliforms, but flagged up by the MinION screening method, and confirmed by a qPCR assay. Plate counting methods remain important to assess viability of faecal coliforms in disinfected water sources. We outline a systematic approach for data collection and interpretation of such complementary results. In the Kathmandu Valley, there is high variability of water quality from different sources, including for treated water samples, illustrating the importance of disinfection at the point of use.

Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations

“…Our HTS data are based on the pyrosequencing 454 method (Roche) that has been replaced by newer platforms that provide sequencing depth orders of magnitude higher, such as Illumina or Oxford Nanopore. However, as the main biases arise from DNA extraction, PCR amplification, and uneven 16S and 18S rRNA gene copy numbers (47), these newer methods will not necessarily improve the quantitative accuracy of the sequencing data, as shown with mock communities sequenced using Illumina (13)(14)(15) and Oxford Nanopore (48,49) platforms. In contrast, the relative abundances of sequences obtained from the same samples by both pyrosequencing and Illumina correlated very strongly (r 2 Ͼ 0.99) (50).…”

A lab in the field: applications of real-time, in situ metagenomic sequencing

“…In this case, both Illumina and nanopore shotgun sequencing revealed comparable abundances of major ARG types. The agreement between the two platforms has been also described for the analysis of different water sources in Nepal through 16S rRNA sequencing [ 60 ]. Although long-reads allowed the classification of 59.41% of the reads down to the species level—no Illumina reads were classified at this level—a significant number of false-positives arose.…”

“…These results were consistent with observations from [ 61 ], which showed that the bacterial identification at the genus level was reliable. Species-level missclassifications could be partially addressed by employing different—and optimized—bioinformatic approaches for the taxonomic classification [ 45 , 62 ], by sequencing the complete 16S-ITS-23S region of the ribosomal operon [ 63 , 64 ], or by coupling MinION sequencing with complementary quantitative PCR assays [ 60 ].…”

De novo species identification using 16S rRNA gene nanopore sequencing

“…Third generation nanopore sequencing has revolutionized the field of analyzing microbial communities, with the promise of on-site high throughput analyses ( Acharya et al, 2019 ). However, despite several recent advances in nanopore sequencing, the error rates are too high for de novo species identification ( Shin et al, 2016 ).…”