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A suitcase laboratory was used for 16S rRNA amplicon sequencing to assess microbial water quality in the holy Bagmati River, Kathmandu, Nepal. SourceTracker analysis and Volcano plots revealed that microbial communities in the downstream part of the river were mainly contributed by untreated sewage. Seasonal variability in the sewage microbiome was reflected in the downstream river water quality. The bacterial genera Acidovorax, Geobacillus and Caulobacter predominated in the upstream sites, while genera containing putative human pathogens and gut bacteria, such as Clostridium, Prevotella, Arcobacter, Lactobacillus, Enterococcus and Streptococcus become prominent in the downstream sites. Marker gene qPCR assays for total bacteria, total coliforms, Human E. coli, Arcobacter butzleri and Vibrio cholerae confirmed the sequencing data trends. Even though basic sanitation provision is nowadays near universal in Nepal, our findings show how inadequate wastewater management may turn an urban river into an open sewer, which poses a public health risk.

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.