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Despite its clinical importance, detection of highly divergent or yet unknown viruses is a major challenge. When human samples are sequenced, conventional alignments classify many assembled contigs as “unknown” since many of the sequences are not similar to known genomes. In this work, we developed ViraMiner, a deep learning-based method to identify viruses in various human biospecimens. ViraMiner contains two branches of Convolutional Neural Networks designed to detect both patterns and pattern-frequencies on raw metagenomics contigs. The training dataset included sequences obtained from 19 metagenomic experiments which were analyzed and labeled by BLAST. The model achieves significantly improved accuracy compared to other machine learning methods for viral genome classification. Using 300 bp contigs ViraMiner achieves 0.923 area under the ROC curve. To our knowledge, this is the first machine learning methodology that can detect the presence of viral sequences among raw metagenomic contigs from diverse human samples. We suggest that the proposed model captures different types of information of genome composition, and can be used as a recommendation system to further investigate sequences labeled as “unknown” by conventional alignment methods. Exploring these highly-divergent viruses, in turn, can enhance our knowledge of infectious causes of diseases. [ABSTRACT FROM AUTHOR]

Highly sensitive and low-cost DNA agarose gel detection systems were developed using non-mutagenic and loading dye-type DNA-staining reagents. The DNA detection system that used Midori Green Direct and Safelook Load-Green, both with an optimum excitation wavelength at ~490 nm, could detect DNA-fragments at the same sensitivity to that of the UV (312 nm)-transilluminator system combined with ethidium bromide, after it was excited by a combination of cyan LED light and a shortpass filter (510 nm). The cyan LED system can be also applied to SYBR Safe that is widely used as a non-toxic dye for post-DNA-staining. Another DNA-detection system excited by black light was also developed. Black light used in this system had a peak emission at 360 nm and caused less damage to DNA due to lower energy of UV rays with longer wavelength when compared to those of short UV rays. Moreover, hardware costs of the black light system were ~$100, less than 1/10 of the commercially available UV (365 nm) transilluminator (>$1,000). EZ-Vision and Safelook Load-White can be used as non-mutagenic and loading dye-type DNA-staining reagents in this system. The black light system had a greater detection sensitivity for DNA fragments stained by EZ-Vision and Safelook Load-White compared with the commercially available imaging system using UV (365 nm) transilluminator. [ABSTRACT FROM AUTHOR]

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely ‘TB Detect’ (containing BioFM-Filter device), ‘TB Concentration and Transport’ (containing Trans-Filter device) and ‘TB DNA Extraction’ kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p<0.05), respectively and an increment in smear grade status (1+ or 2+ to 3+) of 16% over ZN smear and 20% over LED-FM smear. The sensitivity of Mol-DST in presumptive MDR-TB and XDR-TB cases (n = 148) was 90% for Rifampicin (95% confidence interval [CI], 78–96%), 84% for Isoniazid (95% CI, 72–92%), 83% for Fluoroquinolones (95% CI, 66–93%) and 75% for Aminoglycosides (95% CI, 35–97%), using phenotypic DST as the reference standard. Test specificity was 88–93% and concordance was ~89–92% (κ value 0.8–0.9). The patient-friendly kits described here address several of the existing challenges and are designed to provide ‘Universal Access’ to rapid TB diagnosis, including drug-resistant disease. Their utility was demonstrated by application to sputum at 2 sites in India. Our findings pave the way for larger studies in different point-of-care settings, including high-density urban areas and remote geographical locations. [ABSTRACT FROM AUTHOR]

Vesicoureteral reflux (VUR) is a complex, heritable disorder. Genome-wide linkage analyses of families affected by VUR have revealed multiple genomic loci linked to VUR. These loci normally harbor a number of genes whose biologically functional variant is yet to be identified. DNA copy number variations (CNVs) have not been extensively studied at high resolution in VUR patients. In this study, we performed array comparative genomic hybridization (aCGH) on a cohort of patients with a history of both VUR and urinary tract infection (UTI) with the objective of identifying genetic variations responsible for VUR and/or UTI susceptibility. UTI/VUR-associated CNVs were identified by aCGH results from the 192 Randomized Intervention for Children With Vesicoureteral Reflux (RIVUR) patients compared to 683 controls. Rare, large CNVs that are likely pathogenic and lead to VUR development were identified using stringent analysis criteria. Because UTI is a common affliction with multiple risk factors, we utilized standard analysis to identify potential disease-modifying CNVs that can contribute to UTI risk. Gene ontology analysis identified that CNVs in innate immunity and development genes were enriched in RIVUR patients. CNVs affecting innate immune genes may contribute to UTI susceptibility in VUR patients and may provide the first step in assisting clinical medicine in determining adverse outcome risk in children with VUR. [ABSTRACT FROM AUTHOR]

Environmental DNA is increasingly being used for assessing the presence and relative abundance of fish in freshwater, but existing protocols typically rely on filtering large volumes of water which is not always practical. We compared the effects of water volume, filtration type and eDNA extraction procedures in the detection of fish in three freshwater bodies (pond, lake and river) using a short fragment of the 12s rRNA mtDNA gene. Quantification of eDNA capture efficiency after DNA extraction, as well as amplification efficiency, were evaluated by conventional PCR and quantitative PCR. No significant differences on eDNA capture yield were found among freshwater bodies, but increasing water volume had a positive effect on eDNA capture and amplification efficiency. Although highest eDNA capture rates were obtained using 2 L of filtered water, 100 mL syringe filtration in combination with ethanol- sodium acetate precipitation proved to be more practical and increased quantitative PCR amplification efficiency by 6.4%. Our results indicate that such method may be optimal to detect fish species effectively across both lotic and lentic freshwater environments. [ABSTRACT FROM AUTHOR]

The ability to detect founding populations of invasive species or rare species with low number of individuals is important for aquatic ecosystem management. Traditional approaches use historical data, knowledge of the species’ ecology and time-consuming surveys. Within the past decade, environmental DNA (eDNA) has emerged as a powerful additional tracking tool. While much work has been done with animals, comparatively very little has been done with aquatic plants. Here we investigated the transportation and seasonal changes in eDNA concentrations for an invasive aquatic species, Elodea canadensis, in Norway. A specific probe assay was developed using chloroplast DNA to study the fate of the targeted eDNA through space and time. The spatial study used a known source of Elodea canadensis within Lake Nordbytjern 400 m away from the lake outlet flowing into the stream Tveia. The rate of disappearance of E. canadensis eDNA was an order of magnitude loss over about 230 m in the lake and 1550 m in the stream. The time series study was performed monthly from May to October in lake Steinsfjorden harbouring E. canadensis, showing that eDNA concentrations varied by up to three orders of magnitude, peaking during fall. In both studies, the presence of suspended clay or turbidity for some samples did not hamper eDNA analysis. This study shows how efficient eDNA tools may be for tracking aquatic plants in the environment and provides key spatial and temporal information on the fate of eDNA. [ABSTRACT FROM AUTHOR]

Given limited resources for managing invasive species, traditional survey methods may not be feasible to implement at a regional scale. Environmental DNA (eDNA) sampling has proven to be an effective method for detecting some invasive species, but comparisons between the detection probability of eDNA and traditional survey methods using modern occupancy modeling methods are rare. We developed a qPCR assay to detect two species of watersnake (Nerodia fasciata and Nerodia sipedon) introduced to California, USA, and we compared the efficacy of eDNA and aquatic trapping. We tested 3–9 water samples each from 30 sites near the known range of N. fasciata, and 61 sites near the known range of N. sipedon. We also deployed aquatic funnel traps at a subset of sites for each species. We detected N. fasciata eDNA in three of nine water samples from just one site, but captured N. fasciata in traps at three of ten sites. We detected N. sipedon eDNA in five of six water samples from one site, which was also the only site of nine at which this species was captured in traps. Traditional trapping surveys had a higher probability of detecting watersnakes than eDNA surveys, and both survey methods had higher detection probability for N. sipedon than N. fasciata. Occupancy models that integrated both trapping and eDNA surveys estimated that 5 sites (95% Credible Interval: 4–10) of 91 were occupied by watersnakes (both species combined), although snakes were only detected at four sites (three for N. fasciata, one for N. sipedon). Our study shows that despite the many successes of eDNA surveys, traditional sampling methods can have higher detection probability for some species. We recommend those tasked with managing species invasions explicitly compare eDNA and traditional survey methods in an occupancy framework to inform their choice of the best method for detecting nascent populations. [ABSTRACT FROM AUTHOR]

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to–(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter. [ABSTRACT FROM AUTHOR]

Fecal pollution management remains one of the biggest challenges for water quality authorities worldwide. Advanced fecal pollution source identification technologies are now available that can provide quantitative information from many animal groups. As public interest in these methodologies grows, it is vital to use standardized procedures with clearly defined data acceptance metrics and conduct field studies demonstrating the use of these techniques to help resolve real-world water quality challenges. Here we apply recently standardized human-associated qPCR methods with custom data acceptance metrics (HF183/BacR287 and HumM2), along with established procedures for ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution sources to (i) demonstrate the feasibility of implementing standardized qPCR procedures in a large-scale field study, and (ii) characterize trends in fecal pollution sources in the research area. A total of 602 water samples were collected over a one-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries in the Tillamook Bay Watershed (OR, USA). Host-associated qPCR results were combined with high-resolution geographic information system (GIS) land use and general indicator bacteria (E. coli) measurements to elucidate water quality fecal pollution trends. Results demonstrate the feasibility of implementing standardized fecal source identification qPCR methods with established data acceptance metrics in a large-scale field study leading to new investigative leads suggesting that elevated E. coli levels may be linked to specific pollution sources and land use activities in the Tillamook Bay Watershed. [ABSTRACT FROM AUTHOR]

The importance of planktonic microbial communities is well acknowledged, since they are fundamental for several natural processes of aquatic ecosystems. Microorganisms naturally control the flux of nutrients, and also degrade and recycle anthropogenic organic and inorganic contaminants. Nevertheless, climate change effects and/or the runoff of nutrients/pollutants can affect the equilibrium of natural microbial communities influencing the occurrence of microbial pathogens and/or microbial toxin producers, which can compromise ecosystem environmental status. Therefore, improved microbial plankton monitoring is essential to better understand how these communities respond to environmental shifts. The study of marine microbial communities typically involves highly cost and time-consuming sampling procedures, which can limit the frequency of sampling and data availability. In this context, we developed and validated an in situ autonomous biosampler (IS-ABS) able to collect/concentrate in situ planktonic communities of different size fractions (targeting prokaryotes and unicellular eukaryotes) for posterior genomic, metagenomic, and/or transcriptomic analysis at a home laboratory. The IS-ABS field prototype is a small size and compact system able to operate up to 150 m depth. Water is pumped by a micropump (TCS MG2000) through a hydraulic circuit that allows in situ filtration of environmental water in one or more Sterivex filters placed in a filter cartridge. The IS-ABS also includes an application to program sampling definitions, allowing pre-setting configuration of the sampling. The efficiency of the IS-ABS was tested against traditional laboratory filtration standardized protocols. Results showed a good performance in terms of DNA recovery, as well as prokaryotic (16S rDNA) and eukaryotic (18S rDNA) community diversity analysis, using either methodologies. The IS-ABS automates the process of collecting environmental DNA, and is suitable for integration in water observation systems, what will contribute to substantially increase biological surveillances. Also, the use of highly sensitive genomic approaches allows a further study of the diversity and functions of whole or specific microbial communities. [ABSTRACT FROM AUTHOR]

Multiscale signal processing techniques such as wavelet filtering have proved to be particularly successful in predicting exon sequences. Traditional wavelet predictor is domain filtering, and enforces exon features by weighting nucleotide values with coefficients. Such a measure performs linear filtering and is not suitable for preserving the short coding exons and the exon-intron boundaries. This paper describes a prediction framework that is capable of non-linearly processing DNA sequences while achieving high prediction rates. There are two key contributions. The first is the introduction of a genomic-inspired multiscale bilateral filtering (MSBF) which exploits both weighting coefficients in the spatial domain and nucleotide similarity in the range. Similarly to wavelet transform, the MSBF is also defined as a weighted sum of nucleotides. The difference is that the MSBF takes into account the variation of nucleotides at a specific codon position. The second contribution is the exploitation of inter-scale correlation in MSBF domain to find the inter-scale dependency on the differences between the exon signal and the background noise. This favourite property is used to sharp the important structures while weakening noise. Three benchmark data sets have been used in the evaluation of considered methods. By comparison with four existing techniques, the prediction results demonstrate that: the proposed method reveals at least improvement of 4.1%, 50.5%, 25.6%, 2.5%, 10.8%, 15.5%, 11.1%, 12.3%, 9.2% and 2.4% on the exons length of 1–24, 25–49, 50–74, 75–99, 100–124, 125–149, 150–174, 175–199, 200–299 and 300–300+, respectively. The MSBF of its nonlinear nature is good at energy compaction, which makes it capable of locating the sharp variations around short exons. The direct scale multiplication of coefficients at several adjacent scales obviously enhanced exon features while the noise contents were suppressed. We show that the non-linear nature and correlation-based property achieved in proposed predictor is greater than that for traditional filtering, which leads to better exon prediction performance. There are some possible applications of this predictor. Its good localization and protection of sharp variations will make the predictor be suitable to perform fault diagnosis of aero-engine. [ABSTRACT FROM AUTHOR]

The coastal tailed frog (Ascaphus truei) is endemic to the Pacific Northwest of North America and is listed as a species of Special Concern under the Canadian Species at Risk Act. Its range is limited to British Columbia where it occurs widely west of the Coast Mountain Ranges extending north almost to the Alaskan Panhandle. The present study focused on surveying within the Cayoosh, Bridge (Shulaps), Seton, Anderson, Carpenter, and Downton Lake drainages. Four years of previous inventory efforts using conventional time-constrained search (TCS) methods detected tailed frog at 23/292 discrete sites (7.9% detection rate) in seven watersheds. Non-invasive environmental DNA (eDNA) methods hold promise for cryptic and low-abundance species detection. We rigorously validated a quantitative real-time polymerase chain reaction (qPCR)-based tool for detecting coastal tailed frog eDNA in water samples. This eASTR4 test is highly specific and sensitive. We applied a two-step targeted eDNA analysis approach on duplicate filtered water samples from a total of 72 sites collected over five days. The first IntegritE-DNA step mitigates false negative results and tests all DNA samples for the ability to support amplification from endogenous plant chloroplast DNA as a measure of sample viability. Three DNA samples failed this step even after inhibitor clean up suggesting that these samples were poor quality and not reliable for targeted species’ DNA analyses. All other DNA samples were deemed viable and were then tested for species-specific DNA. Coastal tailed frog eDNA was detected in 55/72 (76%) discrete stream reaches; nine sites with historical known occurrence were all eDNA positive. The false negative rate for TCS compared to eDNA methods was 58%. The results expand known coastal tailed frog distribution to 24 watersheds effectively more than tripling extant occurrences and confirm a previously suspected, apparently isolated coastal tailed frog metapopulation in the Shulaps drainage. [ABSTRACT FROM AUTHOR]

In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips. [ABSTRACT FROM AUTHOR]

The use of environmental DNA (eDNA) methods for community analysis has recently been developed. High-throughput parallel DNA sequencing (HTS), called eDNA metabarcoding, has been increasingly used in eDNA studies to examine multiple species. However, eDNA metabarcoding methodology requires validation based on traditional methods in all natural ecosystems before a reliable method can be established. To date, relatively few studies have performed eDNA metabarcoding of fishes in aquatic environments where fish communities were intensively surveyed using multiple traditional methods. Here, we have compared fish communities’ data from eDNA metabarcoding with seven conventional multiple capture methods in 31 backwater lakes in Hokkaido, Japan. We found that capture and field surveys of fishes were often interrupted by macrophytes and muddy sediments in the 31 lakes. We sampled 1 L of the surface water and analyzed eDNA using HTS. We also surveyed the fish communities using seven different capture methods, including various types of nets and electrofishing. At some sites, we could not detect any eDNA, presumably because of the polymerase chain reaction (PCR) inhibition. We also detected the marine fish species as sewage-derived eDNA. Comparisons of eDNA metabarcoding and capture methods showed that the detected fish communities were similar between the two methods, with an overlap of 70%. Thus, our study suggests that to detect fish communities in backwater lakes, the performance of eDNA metabarcoding with the use of 1 L surface water sampling is similar to that of capturing methods. Therefore, eDNA metabarcoding can be used for fish community analysis but environmental factors that can cause PCR inhibition, should be considered in eDNA applications. [ABSTRACT FROM AUTHOR]

We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented. [ABSTRACT FROM AUTHOR]

Introduction: Ventilation system filters process recirculated indoor air along with outdoor air. This function inspires the idea of using the filter as an indoor bioaerosol sampler. While promising, there remains a need to investigate several factors that could limit the accuracy of such a sampling approach. Among the important factors are the dynamics of microbial assemblages on filter surfaces over time and the differential influence of outdoor versus recirculated indoor air. Methods: This study collected ventilation system filter samples from an air handling unit on a regular schedule over a 21-week period and analyzed the accumulation patterns of biological particles on the filter both quantitatively (using fluorometry and qPCR) and in terms of microbial diversity (using 16S rDNA and ITS sequencing). Results: The quantitative result showed that total and bacterial DNA accumulated monotonically, rising to 41 ng/cm2 for total DNA and to 2.8 ng/cm2 for bacterial DNA over the 21-week period. The accumulation rate of bacterial DNA correlated with indoor occupancy level. Fungal DNA first rose to 4.0 ng/cm2 before showing a dip to 1.4 ng/cm2 between weeks 6 and 10. The dip indicated a possible artifact of this sampling approach for quantitative analysis as DNA may not be conserved on the filter over the months-long service period. The sequencing results indicate major contributions from outdoor air for fungi and from recirculated indoor air for bacteria. Despite the quantitative changes, the community structure of the microbial assemblages was stable throughout the 21-week sampling period, highlighting the robustness of this sampling method for microbial profiling. Conclusion: This study supports the use of ventilation system filters as indoor bioaerosol samplers, but with caveats: 1) an outdoor reference is required to properly understand the contribution of outdoor bioaerosols; and 2) there is a need to better understand the persistence and durability of the targeted organisms on ventilation system filters. [ABSTRACT FROM AUTHOR]

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent ‘target’ cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact. [ABSTRACT FROM AUTHOR]

Metabarcoding of lake sediments have been shown to reveal current and past biodiversity, but little is known about the degree to which taxa growing in the vegetation are represented in environmental DNA (eDNA) records. We analysed composition of lake and catchment vegetation and vascular plant eDNA at 11 lakes in northern Norway. Out of 489 records of taxa growing within 2 m from the lake shore, 17–49% (mean 31%) of the identifiable taxa recorded were detected with eDNA. Of the 217 eDNA records of 47 plant taxa in the 11 lakes, 73% and 12% matched taxa recorded in vegetation surveys within 2 m and up to about 50 m away from the lakeshore, respectively, whereas 16% were not recorded in the vegetation surveys of the same lake. The latter include taxa likely overlooked in the vegetation surveys or growing outside the survey area. The percentages detected were 61, 47, 25, and 15 for dominant, common, scattered, and rare taxa, respectively. Similar numbers for aquatic plants were 88, 88, 33 and 62%, respectively. Detection rate and taxonomic resolution varied among plant families and functional groups with good detection of e.g. Ericaceae, Roseaceae, deciduous trees, ferns, club mosses and aquatics. The representation of terrestrial taxa in eDNA depends on both their distance from the sampling site and their abundance and is sufficient for recording vegetation types. For aquatic vegetation, eDNA may be comparable with, or even superior to, in-lake vegetation surveys and may therefore be used as an tool for biomonitoring. For reconstruction of terrestrial vegetation, technical improvements and more intensive sampling is needed to detect a higher proportion of rare taxa although DNA of some taxa may never reach the lake sediments due to taphonomical constrains. Nevertheless, eDNA performs similar to conventional methods of pollen and macrofossil analyses and may therefore be an important tool for reconstruction of past vegetation. [ABSTRACT FROM AUTHOR]

Abstract: The use of environmental DNA (eDNA) as a species detection tool is attracting attention from both scientific and applied fields, especially for detecting invasive or rare species. In order to use eDNA as an efficient and reliable tool, however, we need to understand its origin and state as well as factors affecting its degradation. Various biotic and abiotic environmental factors have been proposed to affect degradation of eDNA in aquatic environments and thus to influence detection rates of species. Here, we were interested in two of them, namely UV light, which can break down DNA, and the presence of filter feeders, which can remove DNA and DNA-bound particles. A few, mostly laboratory-based studies have found minor effects of UVB on the degradation of eDNA. Ultraviolet A radiation (UVA), however, has been neglected although it also causes DNA lesions and is 10- to 100-fold more prevalent than UVB when reaching the earth’s surface. Filter feeders are common in aquatic ecosystem, but their effects on eDNA has hitherto been ignored. We conducted a full-factorial aquatic mesocosm experiment under near-natural outdoor conditions manipulating UV radiation as well as the presence of Dreissena polymorpha, a strong filter feeder capable of filtering cells or organelles containing DNA. Surprisingly, we found that neither UV radiation nor the presence of the filter feeder affected eDNA-based detection rates of macroinvertebrates, even though the experiment took place in summer when UV radiation intensity and filtration activity is high for the chosen experimental site and conditions. These results, in combination with studies from marine or laboratory settings finding no effect of sunlight and its UV components on the detectability of eDNA, suggest that eDNA based species assessments could be relatively robust with respect to our two factors studied. [ABSTRACT FROM AUTHOR]

Hadal trenches, oceanic locations deeper than 6,000 m, are thought to have distinct microbial communities compared to those at shallower depths due to high hydrostatic pressures, topographical funneling of organic matter, and biogeographical isolation. Here we evaluate the hypothesis that hadal trenches contain unique microbial biodiversity through analyses of the communities present in the bottom waters of the Kermadec and Mariana trenches. Estimates of microbial protein production indicate active populations under in situ hydrostatic pressures and increasing adaptation to pressure with depth. Depth, trench of collection, and size fraction are important drivers of microbial community structure. Many putative hadal bathytypes, such as members related to the Marinimicrobia, Rhodobacteraceae, Rhodospirilliceae, and Aquibacter, are similar to members identified in other trenches. Most of the differences between the two trench microbiomes consists of taxa belonging to the Gammaproteobacteria whose distributions extend throughout the water column. Growth and survival estimates of representative isolates of these taxa under deep-sea conditions suggest that some members may descend from shallower depths and exist as a potentially inactive fraction of the hadal zone. We conclude that the distinct pelagic communities residing in these two trenches, and perhaps by extension other trenches, reflect both cosmopolitan hadal bathytypes and ubiquitous genera found throughout the water column. [ABSTRACT FROM AUTHOR]

The accuracy of next-generation sequencing (NGS) for detecting tumor-specific mutations in plasma DNA is hindered by errors introduced during PCR/sequencing, base substitutions caused by DNA damage, and pre-existing mutations in normal cells that are present at a low frequency. Here, we performed NGS of genes related to pancreatic cancer (comprising 2.8 kb of genomic DNA) in plasma DNA (average 4.5 ng) using molecular barcodes. The average number of sequenced molecules was 900, and the sequencing depth per molecule was 100 or more. We also developed a bioinformatic variant filter, called CV78, to remove variants that were not considered to be tumor-specific, i.e., those that are either absent or occur at low frequencies in the Catalogue of Somatic Mutations in Cancer database. In a cohort comprising 57 pancreatic cancer patients and 12 healthy individuals, sequencing initially identified variants in 31 (54%) and 5 (42%), respectively, whereas after applying the CV78 filter, 19 (33%) and zero were variant-positive. In a validation cohort consisting of 86 patients with pancreatic cancer and 20 patients with intraductal papillary mucinous neoplasm (IPMN), 62 (72%) with pancreatic cancer patients and 10 (50%) IPMN patients were initially variant positive. After CV78 filtering, these values were reduced to 32 (37%) and 1 (5%), respectively. The variant allele frequency of filtered variants in plasma ranged from 0.25% to 76.1%. Therefore, combining NGS and molecular barcodes with subsequent filtering is likely to eliminate most non-tumor-specific mutations. [ABSTRACT FROM AUTHOR]

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. [ABSTRACT FROM AUTHOR]

The use of environmental DNA (eDNA) to determine the presence and distribution of aquatic organisms has become an important tool to monitor and investigate freshwater communities. The successful application of this method in the field, however, is dependent on the effectiveness of positive DNA verification, which is influenced by site-specific environmental parameters. Factors affecting eDNA concentrations in aquatic ecosystems include flow conditions, and the presence of substances that possess DNA-binding properties or inhibitory effects. In this study we investigated the influence of different environmental parameters on the detection success of eDNA using the invasive goby Neogobius melanostomus. In a standardized laboratory setup, different conditions of flow, sediment-properties, and fish density were compared, as well as different potential natural inhibitors such as algae, humic substances, and suspended sediment particles. The presence of sediment was mainly responsible for lower eDNA detection in the water samples, regardless of flow-through or standing water conditions and a delayed release of eDNA was detected in the presence of sediment. Humic substances had the highest inhibitory effect on eDNA detection followed by algae and siliceous sediment particles. The results of our study highlight that a successful application of eDNA methods in field surveys strongly depends on site-specific conditions, such as water flow conditions, sediment composition, and suspended particles. All these factors should be carefully considered when sampling, analyzing, and interpreting eDNA detection results. [ABSTRACT FROM AUTHOR]

Genotyping-by-sequencing (GBS) was conducted on 333 Cercospora isolates collected from Beta vulgaris (sugar beet, table beet and swiss chard) in the USA and Europe. Cercospora beticola was confirmed as the species predominantly isolated from leaves with Cercospora leaf spot (CLS) symptoms. However, C. cf. flagellaris also was detected at a frequency of 3% in two table beet fields in New York. Resolution of the spatial structure and identification of clonal lineages in C. beticola populations using genome-wide single nucleotide polymorphisms (SNPs) obtained from GBS was compared to genotyping using microsatellites. Varying distance thresholds (bitwise distance = 0, 1.854599 × 10−4, and 1.298 × 10−3) were used for delineation of clonal lineages in C. beticola populations. Results supported previous reports of long distance dispersal of C. beticola through genotype flow. The GBS-SNP data set provided higher resolution in discriminating clonal lineages; however, genotype identification was impacted by filtering parameters and the distance threshold at which the multi-locus genotypes (MLGs) were contracted to multi-locus lineages. The type of marker or different filtering strategies did not impact estimates of population differentiation and structure. Results emphasize the importance of robust filtering strategies and designation of distance thresholds for delineating clonal lineages in population genomics analyses that depend on individual assignment and identification of clonal lineages. Detection of recurrent clonal lineages shared between the USA and Europe, even in the relaxed-filtered SNP data set and with a conservative distance threshold for contraction of MLGs, provided strong evidence for global genotype flow in C. beticola populations. The implications of intercontinental migration in C. beticola populations for CLS management are discussed. [ABSTRACT FROM AUTHOR]

Introduction: Biological particles deposit on air handling system filters as they process air. This study reports and interprets abundance and diversity information regarding biomass accumulation on ordinarily used filters acquired from several locations in a university environment. Methods: DNA-based analysis was applied both to quantify (via DNA fluorometry and qPCR) and to characterize (via high-throughput sequencing) the microbial material on filters, which mainly processed recirculated indoor air. Results were interpreted in relation to building occupancy and ventilation system operational parameters. Results: Based on accumulated biomass, average DNA concentrations per AHU filter surface area across nine indoor locations after twelve weeks of filter use were in the respective ranges 1.1 to 41 ng per cm2 for total DNA, 0.02 to 3.3 ng per cm2 for bacterial DNA and 0.2 to 2.0 ng DNA per cm2 for fungal DNA. The most abundant genera detected on the AHU filter samples were Clostridium, Streptophyta, Bacillus, Acinetobacter and Ktedonobacter for bacteria and Aspergillus, Cladosporium, Nigrospora, Rigidoporus and Lentinus for fungi. Conditional indoor airborne DNA concentrations (median (range)) were estimated to be 13 (2.6–107) pg/m3 for total DNA, 0.4 (0.05–8.4) pg/m3 for bacterial DNA and 2.3 (1.0–5.1) pg/m3 for fungal DNA. Conclusion: Conditional airborne concentrations and the relative abundances of selected groups of genera correlate well with occupancy level. Bacterial DNA was found to be more responsive than fungal DNA to differences in occupancy level and indoor environmental conditions. [ABSTRACT FROM AUTHOR]

Although human occupancy is a source of airborne bacteria, the role of walkers on bacterial communities in built environments is poorly understood. Therefore, we visualized the impact of walker occupancy combined with other factors (temperature, humidity, atmospheric pressure, dust particles) on airborne bacterial features in the Sapporo underground pedestrian space in Sapporo, Japan. Air samples (n = 18; 4,800L/each sample) were collected at 8:00 h to 20:00 h on 3 days (regular sampling) and at early morning / late night (5:50 h to 7:50 h / 22:15 h to 24:45 h) on a day (baseline sampling), and the number of CFUs (colony forming units) OTUs (operational taxonomic units) and other factors were determined. The results revealed that temperature, humidity, and atmospheric pressure changed with weather. The number of walkers increased greatly in the morning and evening on each regular sampling day, although total walker numbers did not differ significantly among regular sampling days. A slight increase in small dust particles (0.3–0.5μm) was observed on the days with higher temperature regardless of regular or baseline sampling. At the period on regular sampling, CFU levels varied irregularly among days, and the OTUs of 22-phylum types were observed, with the majority being from Firmicutes or Proteobacteria (γ-), including Staphylococcus sp. derived from human individuals. The data obtained from regular samplings reveled that although no direct interaction of walker occupancy and airborne CFU and OTU features was observed upon Pearson's correlation analysis, cluster analysis indicated an obvious lineage consisting of walker occupancy, CFU numbers, OTU types, small dust particles, and seasonal factors (including temperature and humidity). Meanwhile, at the period on baseline sampling both walker and CFU numbers were similarly minimal. Taken together, the results revealed a positive correlation of walker occupancy with airborne bacteria that increased with increases in temperature and humidity in the presence of airborne small particles. Moreover, the results indicated that small dust particles at high temperature and humidity may be a crucial factor responsible for stabilizing the bacteria released from walkers in built environments. The findings presented herein advance our knowledge and understanding of the relationship between humans and bacterial communities in built environments, and will help improve public health in urban communities. [ABSTRACT FROM AUTHOR]

Harnessing information encoded in environmental DNA (eDNA) in marine waters has the potential to revolutionize marine biomonitoring. Whether using organism-specific quantitative PCR assays or metabarcoding in conjunction with amplicon sequencing, scientists have illustrated that realistic organism censuses can be inferred from eDNA. The next step is establishing ways to link information obtained from eDNA analyses to actual organism abundance. This is only possible by understanding the processes that control eDNA concentrations. The present study uses mesocosm experiments to study the persistence of eDNA in marine waters and explore the role of sunlight in modulating eDNA persistence. We seeded solute-permeable dialysis bags with water containing indigenous eDNA and suspended them in a large tank containing seawater. Bags were subjected to two treatments: half the bags were suspended near the water surface where they received high doses of sunlight, and half at depth where they received lower doses of sunlight. Bags were destructively sampled over the course of 87 hours. eDNA was extracted from water samples and used as template for a Scomber japonicus qPCR assay and a marine fish-specific 12S rRNA PCR assay. The latter was subsequently sequenced using a metabarcoding approach. S. japonicus eDNA, as measured by qPCR, exhibited first order decay with a rate constant ~0.01 hr -1 with no difference in decay rate constants between the two experimental treatments. eDNA metabarcoding identified 190 organizational taxonomic units (OTUs) assigned to varying taxonomic ranks. There was no difference in marine fish communities as measured by eDNA metabarcoding between the two experimental treatments, but there was an effect of time. Given the differences in UVA and UVB fluence received by the two experimental treatments, we conclude that sunlight is not the main driver of fish eDNA decay in the experiments. However, there are clearly temporal effects that need to be considered when interpreting information obtained using eDNA approaches. [ABSTRACT FROM AUTHOR]

The fungal pathogen Batrachochytrium dendrobatidis (Bd) drives declines and extinctions in amphibian communities. However, not all regions and species are equally affected. Here, we show that association with amphibian aquatic habitat types (bromeliad phytotelmata versus stream) across Central America results in the odds of being threatened by Bd being five times higher in stream microhabitats. This differential threat of Bd was supported in our study by a significantly lower prevalence of Bd in bromeliad-associated amphibian species compared to riparian species in Honduran cloud forests. Evidence that the bromeliad environment is less favorable for Bd transmission is exemplified by significantly less suitable physicochemical conditions and higher abundance of Bd-ingesting micro-eukaryotes present in bromeliad water. These factors may inhibit aquatic Bd zoospore survival and the development of an environmental reservoir of the pathogen. Bromeliad phytotelmata thus may act as environmental refuges from Bd, which contribute to protecting associated amphibian communities against chytridiomycosis-driven amphibian declines that threaten the nearby riparian communities. [ABSTRACT FROM AUTHOR]

For several hundred years freshwater crayfish (Crustacea—Decapoda—Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species. [ABSTRACT FROM AUTHOR]

In optical DNA mapping technologies sequence-specific intensity variations (DNA barcodes) along stretched and stained DNA molecules are produced. These “fingerprints” of the underlying DNA sequence have a resolution of the order one kilobasepairs and the stretching of the DNA molecules are performed by surface adsorption or nano-channel setups. A post-processing challenge for nano-channel based methods, due to local and global random movement of the DNA molecule during imaging, is how to align different time frames in order to produce reproducible time-averaged DNA barcodes. The current solutions to this challenge are computationally rather slow. With high-throughput applications in mind, we here introduce a parameter-free method for filtering a single time frame noisy barcode (snap-shot optical map), measured in a fraction of a second. By using only a single time frame barcode we circumvent the need for post-processing alignment. We demonstrate that our method is successful at providing filtered barcodes which are less noisy and more similar to time averaged barcodes. The method is based on the application of a low-pass filter on a single noisy barcode using the width of the Point Spread Function of the system as a unique, and known, filtering parameter. We find that after applying our method, the Pearson correlation coefficient (a real number in the range from -1 to 1) between the single time-frame barcode and the time average of the aligned kymograph increases significantly, roughly by 0.2 on average. By comparing to a database of more than 3000 theoretical plasmid barcodes we show that the capabilities to identify plasmids is improved by filtering single time-frame barcodes compared to the unfiltered analogues. Since snap-shot experiments and computational time using our method both are less than a second, this study opens up for high throughput optical DNA mapping with improved reproducibility. [ABSTRACT FROM AUTHOR]

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3–5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10–15 m) in Macquarie Harbour (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbour (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was determined for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% saturation) and lower DO (20% saturation) levels, revealing that the eDNA can be detected for 4 and 16 hours respectively, after which eDNA concentration drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concentrations, it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) has recently been used for detecting the distribution of macroorganisms in various aquatic habitats. In this study, we applied an eDNA method to estimate the distribution of the Japanese clawed salamander, Onychodactylus japonicus, in headwater streams. Additionally, we compared the detection of eDNA and hand-capturing methods used for determining the distribution of O. japonicus. For eDNA detection, we designed a qPCR primer/probe set for O. japonicus using the 12S rRNA region. We detected the eDNA of O. japonicus at all sites (with the exception of one), where we also observed them by hand-capturing. Additionally, we detected eDNA at two sites where we were unable to observe individuals using the hand-capturing method. Moreover, we found that eDNA concentrations and detection rates of the two water sampling areas (stream surface and under stones) were not significantly different, although the eDNA concentration in the water under stones was more varied than that on the surface. We, therefore, conclude that eDNA methods could be used to determine the distribution of macroorganisms inhabiting headwater systems by using samples collected from the surface of the water. [ABSTRACT FROM AUTHOR]

The Honghe-Hani landscape in China is a UNESCO World Natural Heritage site due to the beauty of its thousands of rice terraces, but these structures are in danger from the invasive crayfish Procambarus clarkii. Crayfish dig nest holes, which collapse terrace walls and destroy rice production. Under the current control strategy, farmers self-report crayfish and are issued pesticide, but this strategy is not expected to eradicate the crayfish nor to prevent their spread since farmers are not able to detect small numbers of crayfish. Thus, we tested whether environmental DNA (eDNA) from paddy-water samples could provide a sensitive detection method. In an aquarium experiment, Real-time Quantitative polymerase chain reaction (qPCR) successfully detected crayfish, even at a simulated density of one crayfish per average-sized paddy (with one false negative). In a field test, we tested eDNA and bottle traps against direct counts of crayfish. eDNA successfully detected crayfish in all 25 paddies where crayfish were observed and in none of the 7 paddies where crayfish were absent. Bottle-trapping was successful in only 68% of the crayfish-present paddies. eDNA concentrations also correlated positively with crayfish counts. In sum, these results suggest that single samples of eDNA are able to detect small crayfish populations, but not perfectly. Thus, we conclude that a program of repeated eDNA sampling is now feasible and likely reliable for measuring crayfish geographic range and for detecting new invasion fronts in the Honghe Hani landscape, which would inform regional control efforts and help to prevent the further spread of this invasive crayfish. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) is DNA shed by organisms into surrounding environments such as soil and water. The new methods using eDNA as a marker for species detection are being rapidly developed. Here we explore basic knowledge regarding the dependence of the eDNA degradation rate on time and water temperature, and the relationship between eDNA degradation and bacterial abundance. This subject has not been well clarified, even though it is essential for improving the reliability of eDNA analysis. To determine the time- and water temperature-dependent degradation of eDNA, river water was sampled and eDNA concentrations were determined for ayu sweetfish (Plecoglossus altivelis altivelis) and common carp (Cyprinus carpio) at seven time points, over a 48-h period, and at three different water temperatures. The degradation of eDNA was modeled for each species using an existing exponential decay model with an extension to include water temperature effects. The degradation models were constructed for ayu sweetfish as Nt = 229,901.2 × exp [− (0.01062 × k − 0.07081) × t] and for common carp as Nt = 2,558.0 × exp [− (0.01075 × k − 0.07372) × t]. Nt is the DNA concentration at time t (elapsed time in hours) and k is the water temperature (°C). We also measured the concentration of eDNA derived from purified genomic DNA of the common carp, which was spiked into aquarium water without the target species, and we measured the bacterial abundance in the sample water after 12 and 24 h of incubation. Environmental DNA degradation was accelerated at higher water temperatures (generalized linear model, GLM; p < 0.001), but bacterial abundance did not have a significant effect on eDNA degradation (GLM, p = 0.097). These results suggest that the proper treatment of this temperature effect in data interpretations and adjustments would increase the reliability of eDNA analysis in future studies. [ABSTRACT FROM AUTHOR]

The difficulty of censusing marine animal populations hampers effective ocean management. Analyzing water for DNA traces shed by organisms may aid assessment. Here we tested aquatic environmental DNA (eDNA) as an indicator of fish presence in the lower Hudson River estuary. A checklist of local marine fish and their relative abundance was prepared by compiling 12 traditional surveys conducted between 1988–2015. To improve eDNA identification success, 31 specimens representing 18 marine fish species were sequenced for two mitochondrial gene regions, boosting coverage of the 12S eDNA target sequence to 80% of local taxa. We collected 76 one-liter shoreline surface water samples at two contrasting estuary locations over six months beginning in January 2016. eDNA was amplified with vertebrate-specific 12S primers. Bioinformatic analysis of amplified DNA, using a reference library of GenBank and our newly generated 12S sequences, detected most (81%) locally abundant or common species and relatively few (23%) uncommon taxa, and corresponded to seasonal presence and habitat preference as determined by traditional surveys. Approximately 2% of fish reads were commonly consumed species that are rare or absent in local waters, consistent with wastewater input. Freshwater species were rarely detected despite Hudson River inflow. These results support further exploration and suggest eDNA will facilitate fine-scale geographic and temporal mapping of marine fish populations at relatively low cost. [ABSTRACT FROM AUTHOR]

Recent development of environmental DNA (eDNA) analysis allows us to survey underwater macro-organisms easily and cost effectively; however, there have been no reports on eDNA detection or quantification for jellyfish. Here we present the first report on an eDNA analysis of marine jellyfish using Japanese sea nettle (Chrysaora pacifica) as a model species by combining a tank experiment with spatial and temporal distribution surveys. We performed a tank experiment monitoring eDNA concentrations over a range of time intervals after the introduction of jellyfish, and quantified the eDNA concentrations by quantitative real-time PCR. The eDNA concentrations peaked twice, at 1 and 8 h after the beginning of the experiment, and became stable within 48 h. The estimated release rates of the eDNA in jellyfish were higher than the rates previously reported in fishes. A spatial survey was conducted in June 2014 in Maizuru Bay, Kyoto, in which eDNA was collected from surface water and sea floor water samples at 47 sites while jellyfish near surface water were counted on board by eye. The distribution of eDNA in the bay corresponded with the distribution of jellyfish inferred by visual observation, and the eDNA concentration in the bay was ~13 times higher on the sea floor than on the surface. The temporal survey was conducted from March to November 2014, in which jellyfish were counted by eye every morning while eDNA was collected from surface and sea floor water at three sampling points along a pier once a month. The temporal fluctuation pattern of the eDNA concentrations and the numbers of observed individuals were well correlated. We conclude that an eDNA approach is applicable for jellyfish species in the ocean. [ABSTRACT FROM AUTHOR]

Understanding the cell-specific binding patterns of transcription factors (TFs) is fundamental to studying gene regulatory networks in biological systems, for which ChIP-seq not only provides valuable data but is also considered as the gold standard. Despite tremendous efforts from the scientific community to conduct TF ChIP-seq experiments, the available data represent only a limited percentage of ChIP-seq experiments, considering all possible combinations of TFs and cell lines. In this study, we demonstrate a method for accurately predicting cell-specific TF binding for TF-cell line combinations based on only a small fraction (4%) of the combinations using available ChIP-seq data. The proposed model, termed TFImpute, is based on a deep neural network with a multi-task learning setting to borrow information across transcription factors and cell lines. Compared with existing methods, TFImpute achieves comparable accuracy on TF-cell line combinations with ChIP-seq data; moreover, TFImpute achieves better accuracy on TF-cell line combinations without ChIP-seq data. This approach can predict cell line specific enhancer activities in K562 and HepG2 cell lines, as measured by massively parallel reporter assays, and predicts the impact of SNPs on TF binding. [ABSTRACT FROM AUTHOR]

Background: A simple and reliable DNA extraction of hepatitis B virus (HBV) is critical in developing an ultrasensitive detection method for HBV infection. Current commercially available serum Hepatitis B Virus (HBV) DNA extraction methods are time-consuming, expensive and/or require specialized equipment, which hinders wide adoption of clinical laboratories. This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR (qPCR) detection. Methods: Serum proteins were precipitated by phenol to release HBV DNA in the supernatant which was then transferred to the UF devices. The resultant DNA concentrate was eluted and released into qPCR pre-mixture. The UF-qPCR assay performance, including recovery rate, linearity, detection sensitivity, precision and diagnostic accuracy that compared to the CAP-CTM V2.0 assay by analyzing batched low viral load clinical samples was evaluated. Results: The recovery rate of the UF-based HBV DNA extraction method was above 80%. The assay linearity was demonstrated with a slope of 0.95 and R2 values of 0.99. Limit-of-detection (LOD) of the UF-qPCR assay was determined to be 12.1IU/ml. The coefficient of variation (CV) of HBV quantitation for high, low and limit titer samples was 2.28%, 5.77% and 25.59%, respectively. Accuracy of the UF-qPCR assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01). Conclusions: The UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV. [ABSTRACT FROM AUTHOR]

The analysis of environmental DNA (eDNA) is a powerful, non-destructive technique for detecting rare or hard to find freshwater organisms. In this study, we investigated the effectiveness of environmental DNA analysis as a method for detecting a rare amphibian, the golden tree frog (Phytotriades auratus). These frogs are believed to live exclusively within one species of tank bromeliad, Glomeropitcairnia erectiflora, found on the highest peaks of the island of Trinidad in the West Indies. Previous survey methods for this species involved bromeliad destruction, while here we collected and analyzed water samples from discrete pools within G. erectiflora plants for species-specific DNA. We found 1) that we can identify the presence of P. auratus in the bromeliads using environmental DNA analysis, and 2) that environmental DNA evidence indicates the presence of a previously undiscovered P. auratus population, increasing the species’ range from two isolated ‘sky islands’ to three. [ABSTRACT FROM AUTHOR]

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs. [ABSTRACT FROM AUTHOR]

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water. [ABSTRACT FROM AUTHOR]

The spread of Mysis diluviana, a small glacial relict crustacean, outside its native range has led to unintended shifts in the composition of native fish communities throughout western North America. As a result, biologists seek accurate methods of determining the presence of M. diluviana, especially at low densities or during the initial stages of an invasion. Environmental DNA (eDNA) provides one solution for detecting M. diluviana, but building eDNA markers that are both sensitive and species-specific is challenging when the distribution and taxonomy of closely related non-target taxa are poorly understood, published genetic data are sparse, and tissue samples are difficult to obtain. To address these issues, we developed a pair of independent eDNA markers to increase the likelihood of a positive detection of M. diluviana when present and reduce the probability of false positive detections from closely related non-target species. Because tissue samples of closely-related and possibly sympatric, non-target taxa could not be obtained, we used synthetic DNA sequences of closely related non-target species to test the specificity of eDNA markers. Both eDNA markers yielded positive detections from five waterbodies where M. diluviana was known to be present, and no detections in five others where this species was thought to be absent. Daytime samples from varying depths in one waterbody occupied by M. diluviana demonstrated that samples near the lake bottom produced 5 to more than 300 times as many eDNA copies as samples taken at other depths, but all samples tested positive regardless of depth. [ABSTRACT FROM AUTHOR]

Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs. [ABSTRACT FROM AUTHOR]

We describe the sampling of sixty-three uncultured hospital air samples collected over a six-month period and analysis using shotgun metagenomic sequencing. Our primary goals were to determine the longitudinal metagenomic variability of this environment, identify and characterize genomes of potential pathogens and determine whether they are atypical to the hospital airborne metagenome. Air samples were collected from eight locations which included patient wards, the main lobby and outside. The resulting DNA libraries produced 972 million sequences representing 51 gigabases. Hierarchical clustering of samples by the most abundant 50 microbial orders generated three major nodes which primarily clustered by type of location. Because the indoor locations were longitudinally consistent, episodic relative increases in microbial genomic signatures related to the opportunistic pathogens Aspergillus, Penicillium and Stenotrophomonas were identified as outliers at specific locations. Further analysis of microbial reads specific for Stenotrophomonas maltophilia indicated homology to a sequenced multi-drug resistant clinical strain and we observed broad sequence coverage of resistance genes. We demonstrate that a shotgun metagenomic sequencing approach can be used to characterize the resistance determinants of pathogen genomes that are uncharacteristic for an otherwise consistent hospital air microbial metagenomic profile. [ABSTRACT FROM AUTHOR]

Massively parallel sequencing (MPS) combined with bioinformatic analysis has been widely applied to detect fetal chromosomal aneuploidies such as trisomy 21, 18, 13 and sex chromosome aneuploidies (SCAs) by sequencing cell-free fetal DNA (cffDNA) from maternal plasma, so-called non-invasive prenatal testing (NIPT). However, many technical challenges, such as dependency on correct fetal sex prediction, large variations of chromosome Y measurement and high sensitivity to random reads mapping, may result in higher false negative rate (FNR) and false positive rate (FPR) in fetal sex prediction as well as in SCAs detection. Here, we developed an optimized method to improve the accuracy of the current method by filtering out randomly mapped reads in six specific regions of the Y chromosome. The method reduces the FNR and FPR of fetal sex prediction from nearly 1% to 0.01% and 0.06%, respectively and works robustly under conditions of low fetal DNA concentration (1%) in testing and simulation of 92 samples. The optimized method was further confirmed by large scale testing (1590 samples), suggesting that it is reliable and robust enough for clinical testing. [ABSTRACT FROM AUTHOR]

Background: Since the discovery of cell-free foetal DNA in the plasma of pregnant women, many non-invasive prenatal testing assays have been developed. In the area of skeletal dysplasia diagnosis, some PCR-based non-invasive prenatal testing assays have been developed to facilitate the ultrasound diagnosis of skeletal dysplasias that are caused by de novo mutations. However, skeletal dysplasias are a group of heterogeneous genetic diseases, the PCR-based method is hard to detect multiple gene or loci simultaneously, and the diagnosis rate is highly dependent on the accuracy of the ultrasound diagnosis. In this study, we investigated the feasibility of using targeted capture sequencing to detect foetal de novo pathogenic mutations responsible for skeletal dysplasia. Methodology/Principal Findings: Three families whose foetuses were affected by skeletal dysplasia and two control families whose foetuses were affected by other single gene diseases were included in this study. Sixteen genes related to some common lethal skeletal dysplasias were selected for analysis, and probes were designed to capture the coding regions of these genes. Targeted capture sequencing was performed on the maternal plasma DNA, the maternal genomic DNA, and the paternal genomic DNA. The de novo pathogenic variants in the plasma DNA data were identified using a bioinformatical process developed for low frequency mutation detection and a strict variant interpretation strategy. The causal variants could be specifically identified in the plasma, and the results were identical to those obtained by sequencing amniotic fluid samples. Furthermore, a mean of 97% foetal specific alleles, which are alleles that are not shared by maternal genomic DNA and amniotic fluid DNA, were identified successfully in plasma samples. Conclusions/Significance: Our study shows that capture sequencing of maternal plasma DNA can be used to non-invasive detection of de novo pathogenic variants. This method has the potential to be used to facilitate the prenatal diagnosis of skeletal dysplasia. [ABSTRACT FROM AUTHOR]

In a rapidly changing world we need methods to efficiently assess biodiversity in order to monitor ecosystem trends. Ecological monitoring often uses plant community composition to infer quality of sites but conventional aboveground surveys only capture a snapshot of the actively growing plant diversity. Environmental DNA (eDNA) extracted from soil samples, however, can include taxa represented by both active and dormant tissues, seeds, pollen, and detritus. Analysis of this eDNA through DNA metabarcoding provides a more comprehensive view of plant diversity at a site from a single assessment but it is not clear which DNA markers are best used to capture this diversity. Sequence recovery, annotation, and sequence resolution among taxa were evaluated for four established DNA markers (matK, rbcL, ITS2, and the trnL P6 loop) in silico using database sequences and in situ using high throughput sequencing of 35 soil samples from a remote boreal wetland. Overall, ITS2 and rbcL are recommended for DNA metabarcoding of vascular plants from eDNA when not using customized or geographically restricted reference databases. We describe a new framework for evaluating DNA metabarcodes and, contrary to existing assumptions, we found that full length DNA barcode regions could outperform shorter markers for surveying plant diversity from soil samples. By using current DNA barcoding markers rbcL and ITS2 for plant metabarcoding, we can take advantage of existing resources such as the growing DNA barcode database. Our work establishes the value of standard DNA barcodes for soil plant eDNA analysis in ecological investigations and biomonitoring programs and supports the collaborative development of DNA barcoding and metabarcoding. [ABSTRACT FROM AUTHOR]

Recreational water quality is commonly monitored by means of culture based faecal indicator organism (FIOs) assays. However, these methods are costly and time-consuming; a serious disadvantage when combined with issues such as non-specificity and user bias. New culture and molecular methods have been developed to counter these drawbacks. This study compared industry-standard IDEXX methods (Colilert and Enterolert) with three alternative approaches: 1) TECTA™ system for E. coli and enterococci; 2) US EPA’s 1611 method (qPCR based enterococci enumeration); and 3) Next Generation Sequencing (NGS). Water samples (233) were collected from riverine, estuarine and marine environments over the 2014–2015 summer period and analysed by the four methods. The results demonstrated that E. coli and coliform densities, inferred by the IDEXX system, correlated strongly with the TECTA™ system. The TECTA™ system had further advantages in faster turnaround times (~12 hrs from sample receipt to result compared to 24 hrs); no staff time required for interpretation and less user bias (results are automatically calculated, compared to subjective colorimetric decisions). The US EPA Method 1611 qPCR method also showed significant correlation with the IDEXX enterococci method; but had significant disadvantages such as highly technical analysis and higher operational costs (330% of IDEXX). The NGS method demonstrated statistically significant correlations between IDEXX and the proportions of sequences belonging to FIOs, Enterobacteriaceae, and Enterococcaceae. While costs (3,000% of IDEXX) and analysis time (300% of IDEXX) were found to be significant drawbacks of NGS, rapid technological advances in this field will soon see it widely adopted. [ABSTRACT FROM AUTHOR]