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Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

A multiple host-specific PCR assays are used to assess the exposure to feces in two watersheds with different management histories by tracking cattle feces bacterial populations. The results provide practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.

Microbial fate and transport in watersheds should include a microbial source apportionment analysis that estimates the importance of each source, relative to each other and in combination, by capturing their impacts spatially and temporally under various scenarios. A loosely configured software infrastructure was used in microbial source-to-receptor modeling by focusing on animal- and human-impacted mixed-use watersheds. Components include data collection software, a microbial source module that determines loading rates from different sources, a watershed model, an inverse model for calibrating flows and microbial densities, tabular and graphical viewers, software to convert output to different formats, and a model for calculating risk from pathogen exposure. The system automates, as much as possible, the manual process of accessing and retrieving data and completes input data files of the models. The workflow considers land-applied manure from domestic animals on undeveloped areas; direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; pastureland, cropland, forest, and urban or engineered areas; sources that directly release to streams from leaking septic systems; and shedding by domestic animals directly to streams. The infrastructure also considers point sources from regulated discharges. An application is presented on a real-world watershed and helps answer questions such as: What are the major microbial sources? What practices contribute to contamination at the receptor location? What land-use types influence contamination at the receptor location? and Under what conditions do these sources manifest themselves? This research aims to improve our understanding of processes related to pathogen and indicator dynamics in mixed-use watershed systems. [ABSTRACT FROM AUTHOR]

A series of simulated rainfall-runoff experiments with applications of different manure types (cattle solid pats, poultry dry litter, swine slurry) was conducted across four seasons on a field containing 36 plots (0.75 × 2 m each), resulting in 144 rainfall-runoff events. Simulating time-varying release of Escherichia coli, enterococci, and fecal coliforms from manures applied at typical agronomic rates evaluated the efficacy of the Bradford-Schijven model modified by adding terms for release efficiency and transportation loss. Two complementary, parallel approaches were used to calibrate the model and estimate microbial release parameters. The first was a four-step sequential procedure using the inverse model PEST, which provides appropriate initial parameter values. The second utilized a PEST/bootstrap procedure to estimate average parameters across plots, manure age, and microbe, and to provide parameter distributions. The experiment determined that manure age, microbe, and season had no clear relationship to the release curve. Cattle solid pats released microbes at a different, slower rate than did poultry dry litter or swine slurry, which had very similar release patterns. These findings were consistent with other published results for both bench- and field-scale, suggesting the modified Bradford-Schijven model can be applied to microbial release from manure. [ABSTRACT FROM AUTHOR]

Although development of next generation sequencing (NGS) has substantially improved our understanding of the microbial ecology of animal feces, previous studies have mostly focused on freshly excreted feces. There is still limited understanding of the aging process dynamics of fecal microbiomes in intact cowpats exposed to natural environments. Fresh cowpats were sampled at multiple time points for 57 days under field conditions; half the samples were exposed to sunlight (unshaded) while the other half was protected from sunlight (shaded). The 16SRNA hypervariable region 4 was amplified from each sample and sequenced on an Illumina MiSeq Platform. While Clostridia, Bacteroidia, and Sphingobacteria were dominant classes of bacteria in fresh cowpats, Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and Bacilli were the dominant classes by the end of the study, indicating a general shift from anaerobic to aerobic bacterial populations. This change was most likely influenced by the shift from cattle gut (anaerobic) to pasture ground (aerobic). Reduced moisture in cowpats may also contribute to the community shift since air can penetrate the dryer cowpat more easily. Twelve genera consisting pathogenic bacteria were detected, with Mycobacterium, Bacillus, and Clostridium being the most abundant; their combined abundance accounts for 90% of the total pathogenic genera. Taxonomic richness and diversity increased throughout the study for most samples, which could be due to bacteria regrowth and colonization of bacteria from the environment. In contrast to the high taxonomic diversity, the changes of PICRUSt inferred function profile were minimal for all cowpats throughout the study, which suggest that core functions predicted by PICRUSt may be too conserved to distinguish differences between aerobe and anaerobe. To the best of our knowledge, this is the first study demonstrating that cowpat exposure to air and sunlight can cause drastic microbiome changes soon after deposition in natural environments. Our findings offer important insights for future research characterizing the microbiome of feces collected in natural environments and the impact of cattle fecal contamination on water resources. [ABSTRACT FROM AUTHOR]