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37 results for “DNA extraction methods”
All raw data for Fukuzawa, T et al. "Environmental DNA extraction method for a high and stable DNA yield"
<p>The all raw data of quantitative PCR for environmental DNA in Fukuzawa, T et al. "Environmental DNA extraction method for a high and stable DNA yield".</p> <p> </p>
Figure 1. DNA extraction with two different protocols from different noninvasive samples. Lines 1, 3, 5 in Evaluation of methods for molecular sex-typing of three heron species from different DNA sources
Figure 1. DNA extraction with two different protocols from different noninvasive samples. Lines 1, 3, 5, and 7: DNA extraction with commercial kit; Lines 2, 4, 6, and 8: DNA extracted with modified standard protocol. Lines 1–2: eggshells (Grey Heron); lines 3–4: eggshell swabs (Grey Heron); lines 5–6: pin feathers (Purple Heron); lines 7–8: contour feathers (Great Egret); 9: negative control; M: molecular marker.
Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter?
<p>The 8 benthic samples from Mainland France (stream Edian, stream Aire, lake Geneva), Sweden (stream Dåmman, Agricultural stream, lake Båtkåjaure) and Mayotte (stream Dapani, stream Majimbini) were collected by scraping material from the surface of stones, following the French standard (AFNOR 2007) used in routine biomonitoring programs.DNA was extracted from each sample (2 replicates) using five DNA extraction methods, followed by the amplification of a short rbcL DNA barcode (312bp) specific to diatoms. PCR products were then sequenced in one random direction using the Ion Torrent™ Personal Genome Machine® (PGM) System according to the manufacturer’s instructions. The data file contains one fastq file per library sequenced with the raw DNA reads, as provided by the sequencing platform (demultiplexing performed by the sequencing platform). An excel file is also provided to make the link between the fastq file number and the sample information (sample origin, DNA extraction method used, number of raw reads).</p>
Supplementary Table S1 (raw data) of "Filtration extraction method using microfluidic channel for measuring environmental DNA "
<p>Supplementary Table S1 (all raw data) of "Filtration extraction method using microfluidic channel for measuring environmental DNA ". Each data of the validation experiment; Experiment 1-4 was located in different sheets..</p>
Data for: Environmental DNA storage and extraction method affects detectability for multiple aquatic invasive species
<p>Environmental DNA (eDNA) refers to genetic material released by organisms into their surrounding environment. Collecting and identifying eDNA has gained popularity for monitoring and surveillance of aquatic invasive species. Invasive species management is most successful when an invasion is identified early while population size is likely to be low, highlighting the importance of eDNA detection sensitivity. Various factors influence DNA yield recovered from environmental samples. Environmental DNA storage and extraction methods, for example, can be adjusted to maximize DNA yield, thereby improving detectability. In this study, we compared the performance of two eDNA storage and extraction methods in detecting three common aquatic invasive species (<em>Bythotrephes longimanus</em>, <em>Dreissena polymorpha</em>, and <em>Faxonius rusticus</em>) across five natural ecosystems of Minnesota, United States. One method involved storing filters in 95% ethanol (EtOH) and extracting DNA using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), whereas the other method used cetyl trimethylammonium bromide (CTAB) for storage and a phenol–chloroform–isoamyl (PCI) procedure for DNA extraction. We also investigated the effect of DNA extract volume (1 μL relative to 3 μL) in qPCR reactions on eDNA detections for the commercial kit method. The CTAB‐PCI method yielded significantly more positive detections, across all three species, compared to the EtOH‐Qiagen method. Moreover, we found that using 1 μL of DNA extract in qPCR reactions was equally effective as using 3 μL. To improve detections of aquatic invasive species, we recommend that researchers store eDNA sample filters in CTAB or a similar lysis buffer such as Longmire's solution and extract with PCI when feasible, but note that lower extract volumes might be used without negative effect when either increasing technical replicates or repurposing samples for the detection of multiple species.</p>
Data for: Environmental DNA storage and extraction method affects detectability for multiple aquatic invasive species
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Data from: Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: a case study with ant cuticular bacteria
High-throughput sequencing of the 16S rRNA gene has considerably helped revealing the essential role of bacteria living on insect cuticles in the ecophysiology and behavior of their hosts. However, our understanding of host-cuticular microbiota feedbacks remains hampered by the difficulties to working with low bacterial DNA quantities as in individual insect cuticle samples, which are more prone to molecular biases and contaminations. Herein, we conducted a methodological benchmark on the cuticular bacterial loads retrieved from two Neotropical ant species of different body size and ecology: Atta cephalotes (~15 mm) and Pseudomyrmex penetrator (~5 mm). We evaluated the richness and composition of the cuticular microbiota, as well as the amount of biases and contamination produced by four DNA extraction protocols. We also addressed how bacterial communities' characteristics would be affected by the number of individuals or individual body size used for DNA extraction. Most extraction methods yielded similar results in term of bacterial diversity and composition for A. cephalotes (~15 mm). In contrast, greater amounts of artifactual sequences and contaminations, as well as noticeable differences in bacterial communities' characteristics were observed between the extraction methods for P. penetrator (~5 mm). We also found that large (~15 mm) and small (~5 mm) A. cephalotes individuals harbor different bacterial communities. Our benchmark hence suggests that cuticular microbiota of single insect individuals can be reliably retrieved provided that blank controls, appropriate data cleaning, and standardization of individual body size are considered in the experiment.
Evaluation of different methods to extract DNA from serum seeded with methicillin-resistant Staphylococcus aureus
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Optimising recovery of DNA from minimally-invasive sampling methods: efficacy of buccal swabs, preservation strategy and DNA extraction approaches for amphibian studies_Dataset_Rscript
<p>Datasets and Rscript associated with paper draft titled: "<span>Optimising recovery of DNA from minimally-invasive sampling methods: efficacy of buccal swabs, preservation strategy and DNA extraction approaches for amphibian studies".</span></p> <p> </p> <p>Abstract: <span>Studies in evolution, ecology and conservation are increasingly based on genetic and genomic inferences. With increased focus on molecular approaches, ethical concerns about destructive or more invasive techniques need to be considered, with a push for minimally invasive sampling to be optimised. Buccal swabs have been increasingly used to collect DNA in a number of taxa, including amphibians.<span> </span>However, DNA yield and purity from swabs is often low, limiting its use. In this study we compare different types of swabs, preservation method and storage, and DNA extraction technique in three case studies to assess the optimal approach for recovering DNA in anurans. Out of the five different types of swab that we tested, Isohelix MS-02 and Rapidry swabs generated higher DNA yields than other swabs. When comparing storage buffers, ethanol is a better preservative than a non-alcoholic alternative. Dried samples resulted in similar or better final DNA yields than ethanol-fixed samples if kept cool. DNA extraction via a Qiagen</span><span>™</span><span> DNeasy Blood and Tissue Kit and McHale’s salting out extraction method resulted in similar DNA yields but the Qiagen</span><span>™</span><span> kit extracts contained less contamination. We also found that samples produce better DNA recovery if frozen as soon as possible after collection. We provide recommendations for sample collection and extraction under different conditions, including budgetary considerations, size of individual sampled, access to cold storage facilities, and DNA extraction methodology. Maximising efficacy of all of these factors for better DNA recovery will allow buccal swabs to be used for genetic and genomic studies in a range of vertebrates.</span></p>
Data from: Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA
Environmental DNA (eDNA) is used to detect biodiversity by the capture, extraction, and identification of DNA shed to the environment. However, eDNA capture and extraction protocols vary widely across studies. This use of different protocols potentially biases detection results and could significantly hinder a reliable use of eDNA to detect biodiversity. We tested whether choice of eDNA capture and extraction protocols significantly influenced biodiversity detection in aquatic systems. We sampled lake and river water, captured and extracted eDNA using six combinations of different protocols with replication, and tested for the detection of four macroinvertebrate species. Additionally, using the same lake water technical replicates, we compared the effect of capture and extraction protocols on metabarcode detections of biodiversity using 16S for eubacteria and cytochrome c oxidase I (COI) for eukaryotes. Protocol combinations for capture and extraction of eDNA significantly influenced DNA yield and number of sequences obtained from next generation sequencing. We found significantly different detection rates of species ranging from zero percent to thirty-three percent. Differences in which protocol combinations produced the highest metabarcoded biodiversity were detected and demonstrate that different protocols are required for different biodiversity targets. Our results highlight that the choice of molecular protocols used for capture and extraction of eDNA from water can strongly affect biodiversity detection. Consideration of biases caused by choice of protocols should lead to a more consistent and reliable molecular workflow for repeatable and increased detection of biodiversity in aquatic communities.
Data from: HyRAD-X, a versatile method combining exome capture and RAD sequencing to extract genomic information from ancient DNA
Over the last decade, protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been widely developed. Their use is however limited to DNA of relatively high molecular weight. During the last year, several methods exploiting hybridization capture using probes based on RAD-sequencing loci have circumvented this limitation and opened avenues to the study of samples characterized by degraded DNA, such as historical specimens. Here, we present a major update to those methods, namely Hybridization capture from RAD-derived probes obtained from a reduced eXome template (hyRAD-X), a technique applying RAD-sequencing to messenger RNA from one or few fresh specimens to elaborate bench-top produced probes, i.e., a reduced representation of the exome, further used to capture homologous DNA from a samples set. In contrast to previous hybridization-capture methods, the reference catalog on which reads are aligned does not rely on de novo assembly of anonymous RAD-sequencing loci, but on an assembled transcriptome obtained from RNAseq data, thus increasing the accuracy of loci definition and Single-Nucleotide-Polmorphisms (SNP) call, and targeting, specifically, expressed genes. Finally, the capture step of hyRAD-X relies on RNA probes, increasing stringency of hybridization, making it well suited for low-content DNA samples. As a proof of concept, we applied hyRAD-X to subfossil needles from the coniferous tree Abies alba, collected in lake sediments (Origlio, Switzerland) and dating back from 7200-5800 years before present (BP). More specifically we investigated genetic variation before, during, and after an anthropogenic perturbation that caused an abrupt decrease in Abies alba population size, 6500-6200 years BP. HyRAD-X produced a matrix encompassing 524 exome-derived SNPs. Despite a lower observed heterozygosity was observed during the 6.500-6.200 years BP time slice, genetic composition was nearly identical before and after the perturbation, indicating that re-expansion of the population after the decline was driven by autochthonous specimens. To the best of our knowledge, this is the first time a population genomic study incorporating ancient DNA samples of tree subfossils is conducted at a moderate cost using reproducible exome-reduced complexity.
Data from: “Direct PCR” optimization yields a rapid, cost-effective, non-destructive, and efficient method for obtaining DNA barcodes without DNA extraction
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Data from: Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: a case study with ant cuticular bacteria
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Data from: Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA
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Data from: HyRAD-X, a versatile method combining exome capture and RAD sequencing to extract genomic information from ancient DNA
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Utilizing field collected insects for next generation sequencing: effects of sampling, storage, and DNA extraction methods
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Data from: DNA extraction method affects the detection of a fungal pathogen in formalin-fixed specimens using qPCR
Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.
Data from: More than skin and bones: comparing extraction methods and alternative sources of DNA from avian museum specimens
Next-generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol-chloroform and silica column) to three different sources of DNA (toe pad, skin punch, and bone) from ten historical avian museum specimens. We show that a modified phenol-chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol-chloroform protocol, likely a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol-chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.
Supplementary material 1 from: Bernardes SC, von Rintelen T, Alexander S, Lorenz F, von Rintelen K (2023) Assessing 'non-destructive' DNA extraction method in small crustaceans kept in wet collections. Research Ideas and Outcomes 9: e113299. https://doi.org/10.3897/rio.9.e113299
Supplementary Table S1
Supplementary material 3 from: Bernardes SC, von Rintelen T, Alexander S, Lorenz F, von Rintelen K (2023) Assessing 'non-destructive' DNA extraction method in small crustaceans kept in wet collections. Research Ideas and Outcomes 9: e113299. https://doi.org/10.3897/rio.9.e113299
Supplementary Figure S2
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