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.

Figure 1

Results of the experimentally determined composition of the MOCK Community (MC) at (a) family and (b) genus level with MinION and Illumina NGS techniques. The results are based on 16S rRNA amplicon sequencing. Experimentally determined 16S rRNA gene percentage abundance are compared against the actual composition of the MC as reported by the supplier (i.e. Zymo Research). Data points are an average of duplicate samples for Illumina and triplicate samples for MinION sequencing. Error bars indicate the standard deviation.

Figure 2

Molecular microbiology analysis of the MOCK community. Data points are an average of technical triplicate samples and error bars indicate the standard deviation. Genes associated with human E. coli and Vibrio cholerae were not detected.

Figure 3

Map showing the location of the 13 sampling sites in the Kathmandu Valley and types of water sources sampled.

Figure 4

Molecular microbiology analysis of water samples from different water sources in the Kathmandu Valley. Numbers on the x-axis indicate the ID of the different sampling sites as described in Table 1. Data points for qPCR results are an average of technical triplicate samples and error bars indicate the standard deviation. Data points for Illumina NGS sequencing are an average of duplicate samples and error bars indicate the standard deviation. Library preparation for MinION NGS was unsuccessful for the sample from location 10 and thus data are not available (n.a), while the DNA extraction yield for the blank was too low for NGS, and for the sample from location 1, the yield was too low for all molecular microbiology methods.

Figure 5

Result of correlation analysis between different microbial water quality indicators determined with different methods: plate count, qPCR and NGS (A: Illumina and B: MinION). The colour intensities of circles are proportional to the correlation coefficients (Spearman rank correlation, p < 0.05, n = 12), and the circled numbers represents statistically significant coefficients.

Figure 6

Outline of the experimental approach and methods (both traditional and DNA based) used for microbial water quality analysis, and the interpretation of water quality data obtained with those methods. Sample contamination due to non-sterile working practice may also explain discrepancies between different methodological approaches.