Sea lamprey quantitative environmental DNA surveillance

A new version of Visual tool for estimating the fractal dimension of images

Environmental DNA (eDNA) analysis in water, consisting of water sample collection, DNA extraction and analysis, is increasingly important in detecting rare aquatic species and determining their distribution. Optimization of protocols used in an eDNA molecular workflow is critical to reduce its uncertainty in detecting target species, especially rare species. In this study, 250 mL, 500 mL, 1 L and 2 L water samples were collected by filtering method from a pond for Sichuan taimen (Hucho bleekeri) culture, followed by extraction and purification of eDNA using DNeasy Tissue and Blood DNA extraction kit or MoBio Power Water DNA extraction kit. A PCR procedure was then applied using specific primers from the D-loop region of mtDNA in Sichuan taimen. To explore an appropriate eDNA analysis protocol, the effect of filtering method, sample size and extraction protocols on the detection rate of target gene in water eDNA was studied. The results showed that the target gene was 100% detected from the water sample by using DNeasy Tissue and Blood DNA extraction kit, which was much better that the MoBio Power Water DNA extraction kit with a detection rate of 0%. Larger volume of water samples yielded better PCR products, with 2 L being the most appropriate volume. Sequence alignment results showed that target sequence from the D-loop region of mtDNA in Sichuan taimen was successfully amplified. The results indicated that DNA extracting method and water sample volume could strongly affect the detection rate. The protocol combining filtration, 2 L water sample, DNeasy Tissue and Blood DNA extraction kit was appropriate for eDNA analysis, and mtDNA D-loop region was recommended for detecting DNA of Sichuan taimen from water samples.

Cell-based internal standard for qPCR determinations of antibiotic resistance indicators in environmental water samples

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120–150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced.Graphical After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr+][Ntf2-]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH2O double-distilled water, qPCR quantitative PCR

The European weather loach (Misgurnus fossilis) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle (Ct) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis. We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis, total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis.

Biomonitoring programs have evolved beyond the sole use of morphological identification to determine the composition of invertebrate species assemblages in an array of ecosystems. The application of DNA metabarcoding in freshwater systems for assessing benthic invertebrate communities is now being employed to generate biological information for environmental monitoring and assessment. A possible shift from the extraction of DNA from net-collected bulk benthic samples to its extraction directly from water samples for metabarcoding has generated considerable interest based on the assumption that taxon detectability is comparable when using either method. To test this, we studied paired water and benthos samples from a taxon-rich wetland complex, to investigate differences in the detection of arthropod taxa from each sample type. We demonstrate that metabarcoding of DNA extracted directly from water samples is a poor surrogate for DNA extracted from bulk benthic samples, focusing on key bioindicator groups. Our results continue to support the use of bulk benthic samples as a basis for metabarcoding-based biomonitoring, with nearly three times greater total richness in benthic samples compared to water samples. We also demonstrated that few arthropod taxa are shared between collection methods, with a notable lack of key bioindicator EPTO taxa in the water samples. Although species coverage in water could likely be improved through increased sample replication and/or increased sequencing depth, benthic samples remain the most representative, cost-effective method of generating aquatic compositional information via metabarcoding.

To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method. The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed. When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05). The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Fresh potable water is an indispensable drink which humans consume daily in substantial amounts. Nonetheless, very little is known about the composition of the microbial community inhabiting drinking water or its impact on our gut microbiota. In the current study, an exhaustive shotgun metagenomics analysis of the tap water microbiome highlighted the occurrence of a highly genetic biodiversity of the microbial communities residing in fresh water and the existence of a conserved core tap water microbiota largely represented by novel microbial species, representing microbial dark matter. Furthermore, genome reconstruction of this microbial dark matter from water samples unveiled homologous sequences present in the faecal microbiome of humans from various geographical locations. Accordingly, investigation of the faecal microbiota content of a subject that daily consumed tap water for 3 years provides proof for horizontal transmission and colonization of water bacteria in the human gut.

This data set contains the biaxial tension data of mouse skin. It contains 111 total .csv files that provide information on how the data was collected. All data is with respect to the in vitro unloaded reference configuration (see Figure 1 in the description file). The file name provides details on the sample including subject number, sample number, subject age (i.e., young = 12 weeks, aged = 52 weeks), and sample location (i.e., dorsal or ventral). Each data sets contains three .csv files, each labeled and representing one of the three loading configurations – Equibiaxial (1:1 stretch ratio between 11 and 22 direction), OffbiaxialX (1:2 stretch ratio), and OffbiaxialY (2:1 stretch ratio), determined by rake-to-rake displacement in a displacement-controlled test. Every .csv file contains 4 columns. Columns one and two contain the DIC acquired stretches (unitless) of the tissue in the 11 and 22 direction, respectively. Columns three and four contain the Cauchy stresses (in MPa) in the 11 and 22 direction, respectively. Note, the anatomical lateral direction is the 11 direction and anatomical cranial-caudal direction is the 22 direction. These data represent the experimental downstroke data after 5 cycles of preconditioned loading, at a strain rate of approximately 1.25%/s (approximated during testing by rake-to-rake distance), in 37°C 1xPBS. Thickness of tissues for stress calculations were determined via transverse sections in optimal cutting temperature medium post-testing. For more details, we refer the reader to our referenced manuscript.

A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes ≤4 L or >4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes ≤4 L or >4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

ChecklistBank, developed by Catalogue of Life (COL) and the Global Biodiversity Information Facility (GBIF), is a publishing platform and open data repository focused on taxonomic and nomenclatural data sets (checklists). It contains close to 50K datasets, mostly originating from digitised peer reviewed scientific articles mediated by Plazi, amongst others. The COL Checklist (Bánki et al. 2023) is assembled out of a selection of the data sources in ChecklistBank. The Catalogue of Life Checklist is issued with name usage identifiers, as well as a digital object identifier for the Checklist version (with an associated dataset key). The more than 160 data sources that make up the COL Checklist are also issued with digital object identifiers as well as a data set key. The combination of a name usage identifier and the data set key allows for the tracking of names between the various COL Checklist versions. ChecklistBank is built in an open API. It supports data sharing through various exchange formats of the Darwin Core (Darwin Core Task Group 2009) data standard (e.g., Darwin Core-Archives (GBIF 2021) and ColDP), and provides several download and name matching options. The Transforming European Taxonomy through Training, Research, and Innovations (TETTRIs) European Union funded project will contribute to a couple of improvements to ChecklistBank. In the context of the TETTRIs project, a new name usage (i.e., taxon or synonym) matching service against any dataset in ChecklistBank, not just the COL Checklist, was developed. A single name matching service takes query parameters for a single name and optionally its classification. The service allows for bulk matching of names against the ChecklistBank API. This contains the option of matching a classification in a CSV file. The bulk matching allows all names of an entire or a subtree of an existing ChecklistBank dataset to act as the source for names instead of the input matching a CSV file. The bulk matching services are asynchronous and notify a user by email when the results are ready to be downloaded in a CSV file.

CSV is a popular Open Data format widely used in a variety of domains for its simplicity and effectiveness in storing and disseminating data. Unfortunately, data published in this format often does not conform to strict specifications, making automated data extraction from CSV files a painful task. While table discovery from HTML pages or spreadsheets has been studied extensively, extracting tables from CSV files still poses a considerable challenge due to their loosely defined format and limited embedded metadata. In this work we lay out the challenges of discovering tables in CSV files, and propose Pytheas: a principled method for automatically classifying lines in a CSV file and discovering tables within it based on the intuition that tables maintain a coherency of values in each column. We evaluate our methods over two manually annotated data sets: 2000 CSV files sampled from four Canadian Open Data portals, and 2500 additional files sampled from Canadian, US, UK and Australian portals. Our comparison to state-of-the-art approaches shows that Pytheas is able to successfully discover tables with precision and recall of over 95.9% and 95.7% respectively, while current approaches achieve around 89.6% precision and 81.3% recall. Furthermore, Pytheas's accuracy for correctly classifying all lines per CSV file is 95.6%, versus a maximum of 86.9% for compared approaches. Pytheas generalizes well to new data, with a table discovery F-measure above 95% even when trained on Canadian data and applied to data from different countries. Finally, we introduce a confidence measure for table discovery and demonstrate its value for accurately identifying potential errors.

Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples