DNA recovery and identification from stone tool microcracks

The studies described here introduce a model for residue preservation on stone tools. They simulate stone tool manufacture in order to define parameters important for the study of DNA residues. Microscopic examination of stone tools has identified microcracks that trap DNA and protein from animal blood cells. Thorough investigation of different methods to recover residues from stone tools shows that surface washing leaves DNA and protein, trapped in subsurface microcracks. However, other extraction techniques are able to release 60-80% of DNA and protein residues. Previous research documents the identification of protein from stone tools sonicated in 5% ammonium hydroxide, but it remains untested whether the same treatment yields useable DNA. Using this treatment, I identified 13-year-old DNA residues from experimentally manufactured stone tools. In addition, results clearly indicate that washing procedures typically used to curate stone tools removed only a small fraction of the DNA deposited during animal butchery. Twenty-four pieces of chipped stone recovered from the Bugas-Holding site were studied to explore the validity of ancient DNA residue identifications. Nine tools yielded DNA residues. Modern humans did not touch three of these tools, which suggests that the DNA recovered from them was present prior to excavation. One tool, which was handled by excavators without gloves, harbored DNA from three species, and these templates competed during PCR. On at least two tools, handling after excavation introduced animal DNA unrelated to tool use. Careful testing of Bugas-Holding chipped stone suggests that stone tools may harbor both ancient and modern DNA, and that investigators must take great care to exclude modern DNA from ancient specimens. Ultimately, I developed and streamlined a method to analyze DNA-containing residues preserved on stone tools. This led to several technical improvements in ancient DNA residue analysis. These include a more effective DNA recovery protocol, methods to measure sensitivity and inhibition of PCR in each sample, and strategies to surmount competition between templates during amplification, which can occur in samples that contain DNA from multiple species. These new developments will help future investigators achieve the full potential of ancient DNA residue analysis

Identification of specialists and abundance-occupancy relationships among intestinal bacteria of Aves, Mammalia, and Actinopterygii

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 82 (2016): 1496-1503, doi:10.1128/AEM.02456-15.The coalescence of next generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineating host-microbial associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macro-organisms, but it remains unclear how specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA gene amplified from 106 fecal samples spanning Aves, Mammalia, and Actinopterygii (ray-finned fish). The interspecific abundance-occupancy relationship—where widespread taxa tend to be more abundant than narrowly distributed taxa—among operational taxonomic units (OTUs) was investigated within and among host species. In a separate analysis, specialists OTUs that were highly abundant in a single host and rare in all other hosts were identified using a multinomial model without excluding under-sampled OTUs a priori. We also show that intestinal microbes in humans and other vertebrates studied follow a similar interspecific abundance-occupancy relationship compared to plants and animals, as well as microbes in ocean and soil environments; but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest bacterial taxa can be shared among diverse vertebrate hosts in ways similar to those of ‘free-living’ bacteria

Identification of specialists and abundance-occupancy relationships among intestinal bacteria of Aves, Mammalia, and Actinopterygii

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 82 (2016): 1496-1503, doi:10.1128/AEM.02456-15.The coalescence of next generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineating host-microbial associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macro-organisms, but it remains unclear how specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA gene amplified from 106 fecal samples spanning Aves, Mammalia, and Actinopterygii (ray-finned fish). The interspecific abundance-occupancy relationship—where widespread taxa tend to be more abundant than narrowly distributed taxa—among operational taxonomic units (OTUs) was investigated within and among host species. In a separate analysis, specialists OTUs that were highly abundant in a single host and rare in all other hosts were identified using a multinomial model without excluding under-sampled OTUs a priori. We also show that intestinal microbes in humans and other vertebrates studied follow a similar interspecific abundance-occupancy relationship compared to plants and animals, as well as microbes in ocean and soil environments; but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest bacterial taxa can be shared among diverse vertebrate hosts in ways similar to those of ‘free-living’ bacteria

Differential decay of human faecal Bacteroides in marine and freshwater

Genetic markers from Bacteroides and other faecal bacteria are being tested for inclusion in regulations to quantify aquatic faecal contamination and estimate public health risk. For the method to be used quantitatively across environments, persistence and decay of markers must be understood. We measured concentrations of contaminant molecular markers targeting Enterococcus and Bacteroides spp. in marine and freshwater microcosms spiked with human sewage and exposed to either sunlight or dark treatments. We used Bayesian statistics with a delayed Chick-Watson model to estimate kinetic parameters for target decay. DNA-and RNA-based targets decayed at approximately the same rate. Molecular markers persisted (could be detected) longer in marine water. Sunlight increased the decay rates of cultured indicators more than those of molecular markers; sunlight also limited persistence of molecular markers. Within each treatment, Bacteroides markers had similar decay profiles, but some Bacteroides markers significantly differed in decay rates. The role of extracellular DNA in persistence appeared unimportant in the microcosms. Because conditions were controlled, microcosms allowed the effects of specific environmental variables on marker persistence and decay to be measured. While marker decay profiles in more complex environments would be expected to vary from those observed here, the differences we measured suggest that water matrix is an important factor affecting quantitative source tracking and microbial risk assessment applications.Keywords: Propidium monoazide, Microbial ecology, Sunlight inactivation, Quantitative PCR, Real time PCR, Bayesian method, 16S ribosomal RNA, Genetic markers, Bacteria, Growth rateKeywords: Propidium monoazide, Microbial ecology, Sunlight inactivation, Quantitative PCR, Real time PCR, Bayesian method, 16S ribosomal RNA, Genetic markers, Bacteria, Growth rat

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers - Is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.Keywords: Water quality, Microbial source tracking, Fecal pollutio

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark

Have genetic targets for faecal pollution diagnostics and source tracking revolutionised water quality analysis yet?

The impacts on faecal pollution analysis using nucleic acid-based methods, such as PCR and sequencing, in health-related water quality research were assessed by rigorous literature analysis. A wide range of application areas and study designs has been identified since the first application more than 30 years ago (>1,100 publications). Given the consistency of methods and assessment types, we suggest defining this emerging part of science as a new discipline: genetic faecal pollution diagnostics (GFPD) in health-related microbial water quality analysis. Undoubtedly, GFPD has already revolutionised faecal pollution detection and microbial source tracking, the current core applications. GFPD is also expanding to many other research areas, including infection and health risk assessment, evaluation of microbial water treatment, and support of wastewater surveillance. In addition, storage of DNA extracts allows for biobanking, which opens up new perspectives. The tools of GFPD can be combined with cultivation-based standardised faecal indicator enumeration, pathogen detection, and various environmental data types, in an integrated data analysis approach. This comprehensive meta-analysis provides the scientific status quo of this field, including trend analyses and literature statistics, outlining identified application areas, and discussing the benefits and challenges of nucleic acid-based analysis in GFPD

Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.This is the publisher’s final pdf. The published article is copyrighted by the American Society for Microbiology and can be found at: http://aem.asm.org