A High-Throughput DNA-Sequencing Approach for Determining Sources of Fecal Bacteria in a Lake Superior Estuary

Abstract

Current microbial source-tracking (MST) methods, employed to determine sources of fecal contamination in waterways, use molecular markers targeting host-associated bacteria in animal or human feces. However, there is a lack of knowledge about fecal microbiome composition in several animals and imperfect marker specificity and sensitivity. To overcome these issues, a community-based MST method has been developed. Here, we describe a study done in the Lake Superior-Saint Louis River estuary using SourceTracker, a program that calculates the source contribution to an environment. High-throughput DNA sequencing of microbiota from a diverse collection of fecal samples obtained from 11 types of animals (wild, agricultural, and domesticated) and treated effluent (n = 233) was used to generate a fecal library to perform community-based MST. Analysis of 319 fecal and environmental samples revealed that the community compositions in water and fecal samples were significantly different, allowing for the determination of the presence of fecal inputs and identification of specific sources. SourceTracker results indicated that fecal bacterial inputs into the Lake Superior estuary were primarily attributed to wastewater effluent and, to a lesser extent, geese and gull wastes. These results suggest that a community-based MST method may be another useful tool for determining sources of aquatic fecal bacteria.

Fecal pollution: new trends and challenges in microbial source tracking using next‐generation sequencing

“…Due to these limitations, direct DNA sequencing technologies have been suggested for use in MST studies (Brown et al, 2017;Korajkic et al, 2015;Lee et al, 2011;Newton et al, 2013;Unno et al, 2010;. This has been driven, in large part, due to cost reductions of DNA sequencing, the concurrent increase in the number of sequence reads that can be obtained per run and the ability to combine multiple samples (multiplexing) in the same run (Gilbert and Dupont, 2011).…”

“…SourceTracker has recently been used to determine sources of fecal and environmental contamination in coral reefs off the Florida coast, in a Lake Superior estuary and in potable rainwater and recreational beaches in Australia (Ahmed et al, 2015;Brown et al, 2017;Henry et al, 2016;Neave et al, 2014). In these studies, FTLs were prepared by characterising fecal communities in source samples using NGS of hypervariable regions of the 16S rRNA gene.…”

“…Both computational and in vitro experiments were performed to test the accuracy of the qualitative and quantitative predictions generated by SourceTracker, and a previous study found that source predictions for fecal pollution from sources present at high abundance were relatively accurate (Henry et al, 2016). The calculation of the relative standard deviation (RSD) among several runs has been used as a gauge by which to measure confidence (Brown et al, 2017;Henry et al, 2016;McCarthy et al, 2017).…”

Microbial Source Tracking in the Love Creek Watershed, Delaware (USA)

“…human, farm animal, bird, to determine sources of fecal contamination in the environment. The most common MST method uses quantitative real-time polymerase chain reaction (qPCR) and primers that target the 16S rRNA genes of host associated bacteria (Brown et al, 2017; Harwood et al, 2014), however, qPCR may suffer from specificity and sensitivity issues (Brown et al, 2019; Green et al, 2014; Stewart et al, 2013). In the last decade, advancements in high-throughput DNA sequencing has led to large-scale microbial community studies.…”

“…The SourceTracker algorithm has been shown to be more accurate than random forest analysis, or naive Bayesian classification (Knights et al, 2011). SourceTracker has been used to determine contamination sources in the Russian River (Dubinsky et al, 2016), ATM keypads (Bik et al, 2016), recreational beaches in Australia (Ahmed et al, 2015; Brown et al, 2017; Henry et al, 2016; McCarthy et al, 2017), lakes in St. Paul, Minnesota (Brown et al, 2019), and the upper Mississippi River (Staley et al, 2015, 2014).…”