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52 results for “degraded DNA”
How to quantify factors degrading DNA in the environment and predict degradation for effective sampling design
<p>Extra-organismal DNA (eoDNA) from material left behind by organisms (non-invasive DNA: e.g., faeces, hair) or from environmental samples (eDNA: e.g., water, soil) is a valuable source of genetic information. However, the relatively low quality and quantity of eoDNA, which can be further degraded by environmental factors, results in reduced amplification and sequencing success. This is often compensated for through cost- and time-intensive replications of genotyping/sequencing procedures. Therefore, system- and site-specific quantifications of environmental degradation are needed to maximize sampling efficiency (e.g., fewer replicates, shorter sampling durations), and to improve species detection and abundance estimates. Using ten environmentally diverse bat roosts as a case study, we developed a robust modelling pipeline to quantify the environmental factors degrading eoDNA, predict eoDNA quality, and estimate sampling-site-specific ideal exposure duration. Maximum humidity was the strongest eoDNA-degrading factor, followed by exposure duration and then maximum temperature. We also found a positive effect when hottest days occurred later. The strength of this effect fell between the strength of the effects of exposure duration and maximum temperature. With those predictors and information on sampling period (before or after offspring were born), we reliably predicted mean eoDNA quality per sampling visit at new sites with a mean squared error of 0.0349. Site-specific simulations revealed that reducing exposure duration to 2-8 days could substantially improve eoDNA quality for future sampling. Our pipeline identified high humidity and temperature as strong drivers of eoDNA degradation even in the absence of rain and direct sunlight. Furthermore, we outline the pipeline's utility for other systems and study goals, such as estimating sample age, improving eDNA-based species detection, and increasing the accuracy of abundance estimates.</p>
How to quantify factors degrading DNA in the environment and predict degradation for effective sampling design
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Data supporting: Environmental RNA degrades more rapidly than environmental DNA across a broad range of pH conditions
<p>Although the use and development of molecular biomonitoring tools based on environmental nucleic acids (eDNA and eRNA; collectively known as eNAs) have gained broad interest for the quantification of biodiversity in natural ecosystems, studies investigating the impact of site-specific physicochemical parameters on eNA-based detection methods (particularly eRNA) remain scarce. Here, we used a controlled laboratory microcosm experiment to comparatively assess the environmental degradation of eDNA and eRNA across an acid-base gradient following complete removal of the progenitor organism (<em>Daphnia pulex</em>). Using water samples collected over a 30-day period, eDNA and eRNA copy numbers were quantified using a droplet digital PCR (ddPCR) assay targeting the mitochondrial <em>cytochrome c oxidase</em> subunit I (COI) gene of <em>D. pulex</em>. We found that eRNA decayed more rapidly than eDNA at all pH conditions tested, with detectability—predicted by an exponential decay model—for up to 57 hours (eRNA; neutral pH) and 143 days (eDNA; acidic pH) post organismal removal. Decay rates for eDNA were significantly higher in neutral and alkaline conditions than in acidic conditions, while decay rates for eRNA did not differ significantly among pH levels. Collectively, our findings provide the basis for a predictive framework assessing the persistence and degradation dynamics of eRNA and eDNA across a range of ecologically relevant pH conditions, establish the potential for eRNA to be used in spatially and temporally sensitive biomonitoring studies (as it is detectable across a range of pH levels), and may be used to inform future sampling strategies in aquatic habitats.</p>
Fastq sequence files supporting: Assessing the degradation of environmental DNA and RNA based on genomic origin in a metabarcoding context
<p>Molecular tools of species identification based on eNAs (environmental nucleic acids; eDNA and eRNA) have the potential to greatly transform biodiversity science. However, the ability of eNAs to obtain "real-time" biodiversity estimates may be complicated by the differential persistence and degradation dynamics of the molecular template (eDNA or eRNA) and the barcode marker used. Here, we collected water samples over a 28-day period to comparatively assess species detection using eDNA and eRNA metabarcoding of two distinct barcode markers—a mitochondrial mRNA marker (COI) and a nuclear rRNA marker (18S)—following complete removal of <em>Arthropoda </em>taxa in a semi-natural freshwater system. Our findings demonstrate that <em>Arthropoda </em>community composition was largely influenced by marker choice, rather than molecular template, individual microcosm, or sampling time point. Further, although eRNA may capture similar species diversity as the established eDNA method, this finding may be marker dependent. Although we found little to no difference in decay rates observed among sample groups (COI eDNA, COI eRNA, 18S eDNA, 18S eRNA), this result is likely due to limitations in the ability of eNA-based metabarcoding to provide a strong correlation between true eNA copy numbers present in the environment and final read counts obtained (following the metabarcoding workflow). Collectively, our findings provide further support for the use of multi-marker assessments in metabarcoding surveys to unravel the broadest taxonomic diversity possible, highlight the limitations of eNA metabarcoding methods in providing accurate decay rate estimates, as well as establish the need for further comparative studies using both metabarcoding and single-species detection methods to assess the persistence and degradation dynamics of eNAs for a diverse range of taxa.</p>
Data from: Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera)
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Data supporting: Environmental RNA degrades more rapidly than environmental DNA across a broad range of pH conditions
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Fastq sequence files supporting: Assessing the degradation of environmental DNA and RNA based on genomic origin in a metabarcoding context
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Data from: Balancing sample accumulation and DNA degradation rates to optimize noninvasive genetic sampling of sympatric carnivores
Noninvasive genetic sampling, or noninvasive DNA sampling (NDS), can be an effective monitoring approach for elusive, wide-ranging species at low densities. However, few studies have attempted to maximize sampling efficiency. We present a model for combining sample accumulation and DNA degradation to identify the most efficient (i.e. minimal cost per successful sample) NDS temporal design for capture–recapture analyses. We use scat accumulation and faecal DNA degradation rates for two sympatric carnivores, kit fox (Vulpes macrotis) and coyote (Canis latrans) across two seasons (summer and winter) in Utah, USA, to demonstrate implementation of this approach. We estimated scat accumulation rates by clearing and surveying transects for scats. We evaluated mitochondrial (mtDNA) and nuclear (nDNA) DNA amplification success for faecal DNA samples under natural field conditions for 20 fresh scats/species/season from <1–112 days. Mean accumulation rates were nearly three times greater for coyotes (0.076 scats/km/day) than foxes (0.029 scats/km/day) across seasons. Across species and seasons, mtDNA amplification success was ≥95% through day 21. Fox nDNA amplification success was ≥70% through day 21 across seasons. Coyote nDNA success was ≥70% through day 21 in winter, but declined to <50% by day 7 in summer. We identified a common temporal sampling frame of approximately 14 days that allowed species to be monitored simultaneously, further reducing time, survey effort and costs. Our results suggest that when conducting repeated surveys for capture–recapture analyses, overall cost-efficiency for NDS may be improved with a temporal design that balances field and laboratory costs along with deposition and degradation rates.
Data from: Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit
Noninvasive genetic sampling of faecal pellets can be a valuable method for monitoring rare and cryptic wildlife populations, like the pygmy rabbit (Brachylagus idahoensis). To investigate this method's efficiency for pygmy rabbit monitoring, we evaluated the effect of sample age on DNA degradation in faecal pellets under summer field conditions. We placed 275 samples from known individuals in natural field conditions for 1 to 60 days and assessed DNA quality by amplifying a 294 base pair (bp) mitochondrial DNA (mtDNA) locus and 5 nuclear DNA (nDNA) microsatellite loci (111 – 221 bp). DNA degradation was influenced by sample age, DNA type, locus length, and rabbit sex. Both mtDNA and nDNA exhibited high PCR success rates (94.4%) in samples <1 day old. Success rates for microsatellite loci declined rapidly from 80.0% to 42.7% between days 5 and 7, likely due to increased environmental temperature. Success rates for mtDNA amplification remained higher than nDNA over time, with moderate success (66.7%) at 21 days. Allelic dropout rates were relatively high (17.6% at < 1 day) and increased to 100% at 60 days. False allele rates ranged from 0 to 30.0% and increased gradually over time. We recommend collecting samples as fresh as possible for individual identification during summer field conditions. Our study suggests that this method can be useful for future monitoring efforts, including occupancy surveys, individual identification, population estimation, parentage analysis, and monitoring of genetic diversity both of a reintroduced population in central Washington and across their range.
Data from: Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA-based mark-recapture analysis of Sonoran pronghorn
Knowledge of population demographics is important for species management but can be challenging in low-density, wide-ranging species. Population monitoring of the endangered Sonoran pronghorn (Antilocapra americana sonoriensis) is critical for assessing the success of recovery efforts, and noninvasive DNA sampling (NDS) could be more cost-effective and less intrusive than traditional methods. We evaluated faecal pellet deposition rates and faecal DNA degradation rates to maximize sampling efficiency for DNA-based mark–recapture analyses. Deposition data were collected at five watering holes using sampling intervals of 1–7 days and averaged one pellet pile per pronghorn per day. To evaluate nuclear DNA (nDNA) degradation, 20 faecal samples were exposed to local environmental conditions and sampled at eight time points from one to 124 days. Average amplification success rates for six nDNA microsatellite loci were 81% for samples on day one, 63% by day seven, 2% by day 14 and 0% by day 60. We evaluated the efficiency of different sampling intervals (1–10 days) by estimating the number of successful samples, success rate of individual identification and laboratory costs per successful sample. Cost per successful sample increased and success and efficiency declined as the sampling interval increased. Results indicate NDS of faecal pellets is a feasible method for individual identification, population estimation and demographic monitoring of Sonoran pronghorn. We recommend collecting samples >7 days old and estimate that a sampling interval of 4–7 days in summer conditions (i.e. extreme heat and exposure to UV light) will achieve desired sample sizes for mark–recapture analysis while also maximizing efficiency.
Supplementary Table S1and S2 (raw data) of "Effect of salinity and water dilution on environmental DNA degradation in freshwater environments"
<p>All data, including the raw values for the qPCR experiments</p>
DNA-based assessment of environmental degradation in an unknown fauna: the freshwater macroinvertebrates of the Indo-Burmese hotspot
<p>New methods are required for biomonitoring of poorly known tropical ecosystems, but biological assessments of environmental status are limited by insufficient information on taxonomy, composition, and ecology of local communities. The current work applies DNA-based assessment to establish the impact of various types of anthropogenic disturbances on the freshwater macroinvertebrates in an understudied biodiversity hotspot in South Asia, an area that attracts increasing attention for the loss of aquatic ecosystems.</p> <p>We sampled 16 river systems in the Chittagong Hill Tracts region of Bangladesh and characterised habitat intactness based on a set of 14 environmental parameters associated with habitat quality and human activities. Whole-community metabarcoding was used to investigate the distribution of hypothetical species-level clusters (Operational Taxonomic Units, OTUs) across sites of different impacts.</p> <p>We found >900 DNA clusters of insects, decapods and molluscs, dominated by Diptera, which revealed significant variation (p<0.001) in richness across sites. The presumed sensitive Ephemeroptera-Plecoptera-Trichoptera (EPT) represented 15.6% of total OTU richness. The type and strength of anthropogenic stressors varied greatly across streams but did not affect total OTU diversity. In contrast, EPT richness decreased by ~50% in response to habitat degradation. Partial-network analysis revealed 26 OTUs that may serve as potential indicators for either good or poor ecological status. Overall, our results document high diversity, local endemicity and pronounced responses to disturbance in these largely unexplored but threatened habitats.</p> <p><em>Synthesis and applications</em>: The proposed methodology combines local habitat surveys across sites of various degrees of disturbance with species-level metabarcoding, as a model for biological evaluation of water bodies in poorly known and inaccessible places across the world. Implemented here for the Indo-Burmese hotspot, the approach will have great value for applied conservation management as a step towards building a biomonitoring system in this region where currently little is known about the taxonomy, diversity and endemicity in both intact and disturbed ecosystems.</p>
3D printing template for The Water-Exclusion Trap (WET): A 3D printable window trap collector that prevents DNA degradation
<p><span>3D templates of a long-term storable bottom collector for window or pitfall traps. The collection medium in a LEE trap barely evaporates and does not dilute because water is excluded, maximizing DNA preservation. Based on field-collected specimens and lab experiments, we confirm that the LEE significantly outperforms conventional traps in terms of DNA quality. </span></p>
Data from: Enzymatic degradation of liquid droplets of DNA is modulated near the phase boundary
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A quantitative analysis of vertebrate environmental DNA degradation in soil in response to time, UV light and temperature
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Data from: Balancing sample accumulation and DNA degradation rates to optimize noninvasive genetic sampling of sympatric carnivores
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Data from: Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA
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Data from: Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit
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3D printing template for The Water-Exclusion Trap (WET): A 3D printable window trap collector that prevents DNA degradation
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Data from: Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA-based mark-recapture analysis of Sonoran pronghorn
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