Rate Of DNA Degradation Research Articles

Nuclease-induced stepwise photodropping (NISP) to precisely investigate single-stranded DNA degradation behaviors of exonucleases and endonucleases.

Here, we employed a fluorescence-based single molecule method called nuclease-induced stepwise photodropping (NISP) to measure in realtime the DNA degradation mediated by mitochondrial genome maintenance exonuclease 1 (MGME1), a bidirectional single-stranded DNA (ssDNA)-specific exonuclease. The method detects a stepwise decrease in fluorescence signals from Cy3 fluorophores labeled on an immobilized DNA substrate. Using NISP, we successfully determined the DNA degradation rates of 6.3±0.4and 2.0±0.1 nucleotides (nt) s-1 for MGME1 in the 5'-to-3' and 3'-to-5' directions, respectively. These results provide direct evidence of the stronger 5' directionality of MGME1, consistent with its established role in mitochondrial DNA maintenance. Importantly, when we employed NISP to investigate mung bean nuclease, an ss-specific endonuclease, we observed a markedly different NISP pattern, suggesting a distributive cleavage activity of the enzyme. Furthermore, we applied NISP to determine the ssDNA degradation behavior of the double-stranded-specific exonuclease, λexonuclease. These findings underscore the capability of NISP to accurately and reliably measure the degradation of ssDNA by both exo- and endonucleases. Here, we demonstrate NISP as a powerful tool for investigating the ssDNA degradation behavior of nucleases at the single-molecule level.

Environmental DNA dynamics of three species of unionid freshwater mussels

AbstractNorth American freshwater mussels are of special conservation concern due to their high endemism and the multiple anthropogenic stressors affecting them. Of the over 300 species in North America, nearly one third of these species are federally listed as threatened or endangered. Environmental DNA (eDNA) analysis has been successful in detecting freshwater mussels and could aid in monitoring their populations. Production and degradation rates of eDNA for the species of interest are needed to inform interpretation of eDNA detections, allow possible modeling of relative abundance and population location, and aid in mussel conservation through population identification. Here, we designed and tested qPCR assays for three freshwater mussel species, mucket (Ortmanniana ligamentina), fatmucket (Lampsilis siliquoidea), and the federally endangered spectaclecase (Cumberlandia monodonta). We performed laboratory experiments under controlled conditions to measure eDNA shedding and degradation rates for each species. Different biomasses, temperatures, and food regimens were tested independently to determine if these factors influence the amount of DNA produced by the mussels. Degradation rates of eDNA were measured from experimental tank water after mussels were removed. Overall, we observed low eDNA shedding rates for freshwater mussels compared to previous studies of fish eDNA shedding rates. Furthermore, temperature and feeding showed limited or no significant effects in the species studied. Environmental DNA degradation rates were consistent with those reported in the literature for other taxa. Collectively, our results will be useful for designing eDNA monitoring studies, modeling eDNA dispersal, and interpreting eDNA results to help inform freshwater mussel conservation efforts.

Increased ladybird predation and metabolism do not counterbalance increased field aphid population growth under experimental warming

Abstract Climate change may have diverse and complex impacts on species interactions, destabilizing food webs and ecosystem services. The effects of warming on the top‐down biological control of crop pests have been considerably less studied than bottom‐up effects through crop physiological changes. We studied the effect of a 2°C warming in the laboratory and in wheat fields on the predation and metabolism of Harmonia axyridis on wheat aphids using molecular gut content analysis. We also measured the effects of warming on the predation rate and functional response of H. axyridis on each aphid species in the laboratory, as well as on DNA degradation rate. Field densities of Sitobion avenae and Rhopalosiphum padi, the two most abundant wheat aphid species, were increased by 2 and 2.5 times, respectively, under experimental warming, but densities of H. axyridis were not. Field predation rate of H. axyridis on these two aphid was found to be about 25% lower under elevated temperature. This could have been due to faster prey digestion, since degradation of the preferred aphid species, Sitobion avenae, was 1.5 times faster under elevated temperature. However, the functional response of H. axyridis larvae on these two species was 1.5 times higher under warming over the range of prey densities tested (50–250 over 24 h). The total predation rate of H. axyridis larvae on a mixture of S. avenae, R. padi and Schizaphis graminum aphid prey was also increased by 1.4 times, but consumption of R. padi aphids was increased while that of S. graminum was decreased under warming. Overall, our results show that global warming could strongly increase pest outbreaks and destabilize biological pest control, which would likely result in accrued yield losses. Read the free Plain Language Summary for this article on the Journal blog.

Determination of the Genomic DNA Degradation Rate of the Chinese Herb Gentianae crassicaulis Radix During Processing and Storage

Background Accurate identification of Chinese herbal medicines is the basis for their research and utilization. Molecular identification can effectively differentiate original plants from counterfeit plants. The quality of genomic DNA is an important factor affecting molecular identification. However, the processing can lead to DNA degradation of the herbal medicines, which can make it difficult for their molecular identification. Objectives To establish a genomic DNA degradation model of Gentiana crassicaulis Radix to evaluate the effects of processing methods and storage times on genomic DNA integrity. Materials and Methods A genomic DNA degradation model of G. crassicaulis—the original plant source of the Chinese herbal medicine G. crassicaulis Radix—was established using a steam heating method. Genomic DNA integrity of G. crassicaulis Radix was evaluated using capillary electrochromatography (CEC) fingerprinting and polymerase chain reaction (PCR) of DNA barcoding markers following different processing and drying methods, including slicing (sliced roots), no slicing (whole roots), stoving, air drying, and sweating. Results CEC fingerprinting and DNA barcoding PCR effectively evaluated genomic DNA integrity. Compared to whole roots, sliced roots better helped maintain genomic DNA integrity. As the storage time increased, the integrity of the genomic DNA reduced; the integrity of the genomic DNA of sliced roots was greater than that of whole roots. Furthermore, the interactions between slicing and drying methods possibly reduced the genomic DNA integrity. Conclusion A genomic DNA degradation model and an evaluation system for herbal medicines were established. Our findings can help optimize the method for processing G. crassicaulis Radix and establish the traceability of genuine herbal medicines. Key Message The quality of genomic DNA is an important factor affecting molecular identification, which is essential to determine original plants. The results of our study can help develop a method for processing G. crassicaulis Radix and enable the traceability of genuine herbal medicines.

Using eDNA sampling for species-specific fish detection in tropical oceanic samples: limitations and recommendations for future use.

Over the past decade, environmental DNA (eDNA) has become a resourceful tool in conservation and biomonitoring. Environmental DNA has been applied in a variety of environments, but the application to studies of marine fish, particularly at tropical latitudes, are limited. Since many commercially important Caribbean fishes are overexploited, these species are optimal candidates to explore the use of this method as a biomonitoring tool. Specifically, for many of these species, the formation of fish spawning aggregations (FSAs) marks a critical life history event where fishes will gather in large numbers for reproduction. These FSAs are ephemeral in nature, lasting only a few days, but are predictable in time and space which makes them susceptible to overfishing. In this study, we test the feasibility of using an eDNA sampling approach (water and sediment collection) to detect the presence of known FSAs off the west coast of Puerto Rico, with cytochrome c oxidase subunit 1 (CO1) and 12S rRNA (12S) primers designed to target specific species. A total of 290 eDNA samples were collected and, of those, 206 eDNA samples were processed. All eDNA samples varied in DNA concentration, both between replicates and collection methods. A total of 12 primer sets were developed and tested using traditional PCR and qPCR. Despite validation of primer accuracy and sample collection during known peak spawning times, the use of traditional PCR and qPCR with both molecular markers failed to produce species-specific amplification. Thus, a trial test was conducted using the CO1 primers in which target fish DNA was 'spiked' at various concentrations into the respective eDNA samples to determine the target species DNA concentration limit of detection. Upon successful amplification of the trial, results indicated that eDNA samples were below the detection threshold of our methods, suggesting that the number of fish present at the spawning aggregations was inadequate for single-species detection methods. In addition, elements such as the unavoidable presence of non-target DNA, oceanic environmental conditions, shedding rates of target fish, among other biotic and abiotic factors could have affected DNA persistence and degradation rates at the sites. We provide recommendations for species-specific fish detection in lower latitudes, and suggestions for studies aiming to monitor or detect fish spawning aggregations using eDNA sampling.

Biological Evidence Management at the Crime Scene: An Overview

Crime scene serves as the starting point for forensic science and can generate valuable data that must bemeticulously, methodically, scientifically, and lawfully gathered. If the crime scene is not handled appropriately,it can become misleading and render crucial information useless, leading to an investigation on the wrong path.Forensic science has undergone a revolution thanks to the use of DNA technologies. Many times, due to theinvestigator’s lack of scientific expertise regarding the proper collection, preservation, storage, and transportationof biological evidence, the investigator is unable to acquire accurate DNA analysis results, which reduces the valueof the evidence in court. Purity, quantity, and rate of DNA degradation are just a few of the many variables thatgo into creating a decent DNA profile from a biological sample. We highlight and provide useful strategies andrecommendations to assist medical, forensic, and law enforcement professionals in handling biological evidencesfor DNA analysis to prevent contamination, deterioration, and loss of biological evidence’s value.

Temporal rate of postmortem DNA degradation in archived tissue samples: evidence from liver and muscle

AbstractGuidelines identifying best practices for harvesting tissues that lead to optimal DNA preservation are few but are important curatorial concerns for genetic resource collections. We conducted a temporal study to establish rate of DNA degradation of tissue samples extracted from field-caught museum specimens. Five individuals of Sigmodon hispidus were collected and their liver and muscle tissues were harvested. Each tissue type was sectioned into 15 subsamples, and each was preserved in liquid nitrogen at different time intervals (2, 4, 8, 16, and 32 min; 1, 2, 4, 8, and 16 h; and 1, 2, 4, 8, and 16 days) following death. DNA was extracted using an automated robotic instrument and molecular mass profiles were determined fluorometrically. Postmortem DNA degradation was continuous and dependent on time, but also was significantly affected by differences among individual cotton rats. DNA fragments of ≥10,000 base pairs in length were present in muscle samples across all time intervals, whereas DNA fragments of this size in liver samples were no longer present after 8–16 h postmortem. DNA molecular mass profiles showed that muscle samples retained 80% of their longest fragments (≥10,000 base pairs) until 1 day postmortem, whereas liver samples retained the same percentage only until 8 min after death. Although rates of decay were measured from samples in a laboratory (not field) setting, rates of decay presented here can guide field and museum workers in best practices. Results suggest that opportunistic samples, such as those from roadkill specimens, are more likely to be of use for a variety of molecular methods when muscle is preserved. Considerations of differences in rates of degradation may also guide selection of tissue types housed in genetic resource collections, especially under space-limited circumstances.

First large‐scale quantification study of DNA preservation in insects from natural history collections using genome‐wide sequencing

Abstract Insect declines are a global issue with significant ecological and economic ramifications. Yet, we have a poor understanding of the genomic impact these losses can have. Genome‐wide data from historical specimens have the potential to provide baselines of population genetic measures to study population change, with natural history collections representing large repositories of such specimens. However, an initial challenge in conducting historical DNA data analyses is to understand how molecular preservation varies between specimens. Here, we highlight how Next‐Generation Sequencing methods developed for studying archaeological samples can be applied to determine DNA preservation from only a single leg taken from entomological museum specimens, some of which are more than a century old. An analysis of genome‐wide data from a set of 113 red‐tailed bumblebee Bombus lapidarius specimens, from five British museum collections, was used to quantify DNA preservation over time. Additionally, to improve our analysis and further enable future research, we generated a novel assembly of the red‐tailed bumblebee genome. Our approach shows that museum entomological specimens are comprised of short DNA fragments with mean lengths below 100 base pairs (BP), suggesting a rapid and large‐scale post‐mortem reduction in DNA fragment size. After this initial decline, however, we find a relatively consistent rate of DNA decay in our dataset, and estimate a mean reduction in fragment length of 1.9 bp per decade. The proportion of quality filtered reads mapping to our assembled reference genome was around 50%, and decreased by 1.1% per decade. We demonstrate that historical insects have significant potential to act as sources of DNA to create valuable genetic baselines. The relatively consistent rate of DNA degradation, both across collections and through time, mean that population‐level analyses—for example for conservation or evolutionary studies—are entirely feasible, as long as the degraded nature of DNA is accounted for.

Measuring the process and rate of exogenous DNA degradation during digestion in mice

This study aimed to perform qualitative and quantitative examination of DNA degradation during the digestion process in the mouse gut through PCR, qPCR and short tandem repeat (STR) analysis. Human blood leukocytes were gavaged into the digestive tract in mice. GAPDH, TH01, TPOX and D7S820 genes in the contents of the stomach and small intestine were analyzed with PCR and qPCR at various times pre- and post-gavage. Through STR analysis, 21 human genomic DNA loci were analyzed. The half-life of DNA degradation, and the relationship between the average peak area and digestion time were determined. The PCR results showed bands of amplified genes at pre-gavage (0 min) and post-gavage (40, 80 and 120 min) from the mouse stomach contents, whereas no DNA bands from small intestinal chyme were observed after gavage. The qPCR results revealed a significant decrease in DNA concentrations during 40–120 min in the mouse stomach after gavage. At 120 min, 85.62 ± 8.10% of the DNA was degraded, and the half-life of exogenous DNA degradation in the mouse stomach was 70.50 ± 5.46 min. At various digestion times, almost no target genes were detected in the mouse small intestinal chyme. STR analysis showed a decrease in allele numbers with bowel advancement in the small intestine in mice. The degradation of exogenous DNA was higher in the mouse stomach during the first 2 h, and almost complete degradation was observed within 40 min after entering the small intestine in mice.

Long Term Conservation of DNA at Ambient Temperature. Implications for DNA Data Storage

DNA conservation is central to many applications. This leads to an ever-increasing number of samples which are more and more difficult and costly to store or transport. A way to alleviate this problem is to develop procedures for storing samples at room temperature while maintaining their stability. A variety of commercial systems have been proposed but they fail to completely protect DNA from deleterious factors, mainly water. On the other side, Imagene company has developed a procedure for long-term conservation of biospecimen at room temperature based on the confinement of the samples under an anhydrous and anoxic atmosphere maintained inside hermetic capsules. The procedure has been validated by us and others for purified RNA, and DNA in buffy coat or white blood cells lysates, but a precise determination of purified DNA stability is still lacking. We used the Arrhenius law to determine the DNA degradation rate at room temperature. We found that extrapolation to 25°C gave a degradation rate constant equivalent to about 1 cut/century/100 000 nucleotides, a stability several orders of magnitude larger than the current commercialized processes. Such a stability is fundamental for many applications such as the preservation of very large DNA molecules (particularly interesting in the context of genome sequencing) or oligonucleotides for DNA data storage. Capsules are also well suited for this latter application because of their high capacity. One can calculate that the 64 zettabytes of data produced in 2020 could be stored, standalone, for centuries, in about 20 kg of capsules.

Impact assessment of different DNA extraction methods for non-invasive molecular diagnosis of tegumentary leishmaniasis

The aim of this study was to evaluate two methods of nucleic acid extraction (spin-column-based method – commercial kit and direct boil - DB) from swab sampling compared to biopsy sampling for the diagnosis of tegumentary leishmaniasis (TL), (cutaneous - CL and mucocutaneous - MCL forms). The impact of these nucleic acid extraction protocols on different types of PCR and LAMP techniques were compared regarding nucleic acid quality, molecular assays accuracy, indirect quantitation, and costs. The evaluated patients were 57 TL cases (36 CL and 21 MCL) and 34 non-cases. Swab samples extracted by the DB method showed a higher DNA degradation rate and worse DNA quality in comparison to the commercial kit. Molecular tests performed on biopsy samples showed identical or higher performance in all analysis, as compared to their own performance on swab samples for TL (CL and MCL). However, only the SSU rRNA TaqMan™ RT-PCR test showed a significant difference between the performance of biopsy and swab samples extracted by commercial kit. The kDNA-cPCR coupled with swab extracted by commercial kit showed the highest accuracy (95.6%) for TL diagnosis. The sensitivity of the LAMP-RT 18S method in swab samples extracted with a commercial kit (82.5%) was close to that found in biopsy samples (86%) for TL diagnosis. The DB extraction method presented the lowest cost. The use of swab as a minimally-invasive sampling method, associated with an efficient nucleic acid extraction protocol, may represent a low-cost alternative for the diagnosis of CL and MCL.

Long term conservation of DNA at ambient temperature. Implications for DNA data storage.

DNA conservation is central to many applications. This leads to an ever-increasing number of samples which are more and more difficult and costly to store or transport. A way to alleviate this problem is to develop procedures for storing samples at room temperature while maintaining their stability. A variety of commercial systems have been proposed but they fail to completely protect DNA from deleterious factors, mainly water. On the other side, Imagene company has developed a procedure for long-term conservation of biospecimen at room temperature based on the confinement of the samples under an anhydrous and anoxic atmosphere maintained inside hermetic capsules. The procedure has been validated by us and others for purified RNA, and for DNA in buffy coat or white blood cells lysates, but a precise determination of purified DNA stability is still lacking. We used the Arrhenius law to determine the DNA degradation rate at room temperature. We found that extrapolation to 25°C gave a degradation rate constant equivalent to about 1 cut/century/100 000 nucleotides, a stability several orders of magnitude larger than the current commercialized processes. Such a stability is fundamental for many applications such as the preservation of very large DNA molecules (particularly interesting in the context of genome sequencing) or oligonucleotides for DNA data storage. Capsules are also well suited for this latter application because of their high capacity. One can calculate that the 64 zettabytes of data produced in 2020 could be stored, standalone, for centuries, in about 20 kg of capsules.

Comparison of nuclear DNA yield and STR typing success in Second World War petrous bones and metacarpals III

DNA yield and STR typing success differ among skeletal element types and within individual bones. Consequently, it is necessary to identify the skeletal elements and their intra-skeletal parts that will most likely yield utilizable and informative endogenous DNA for human identification of skeletal remains. The petrous portion of the temporal bone has been shown to be the most suitable skeletal part for sampling archaeological skeletons, and it has also been used successfully in some forensic cases. When all representative bone types were analyzed for three complete Second World War skeletons, metatarsals and metacarpals yielded more DNA than petrous bones (which generated full profiles even if the DNA yield was lower) and, among almost 200 Second World War metatarsals and metacarpals analyzed, metacarpals III were found to be the highest-yielding bones. To further improve the sampling strategy in DNA analysis of aged skeletal remains, a comparison between 26 petrous bones and 30 metacarpals III from Second World War skeletons was made considering intra-bone DNA yield variability. In metacarpals III only epiphyses were sampled, and in petrous bones only the dense part within the otic capsule was used. To exclude the influence of taphonomic issues as much as possible, petrous bones and metacarpals III from a single Second World War mass grave were examined. The difference between petrous bones and metacarpals III was explored by measuring nuclear DNA yield and success of STR typing. After cleaning the samples, full demineralization extraction was used to decalcify 0.5 g of powdered bone. PowerQuant (Promega) was used to determine DNA content and DNA degradation rates, and STR typing was performed using the PowerPlex ESI 17 Fast System (Promega). Metacarpals III produced the same DNA yields and STR typing success as petrous bones with no intra-individual difference observed in concentration of DNA, degradation rate, percentage of successfully amplified alleles, and intensity of electrophoretic signals. Sampling and processing of metacarpal III epiphyses is consequently recommended for genetic identification of highly degraded skeletal remains in routine forensic casework and in buried non-commingled aged skeletal remains whenever metacarpals III are preserved. Metacarpals III are easy to sample and less prone to contamination with modern DNA because no saw is needed for sampling in comparison to the petrous portion of the temporal bone. The data obtained in this study further improve the sampling strategy for genetic identification of Second World War skeletal remains in Slovenia.

Oxidative degradation perturbs physico-chemical properties of hemoglobin in cigarette smokers: a threat to different biomolecules

Context Cigarette smokers develop structural modification in hemoglobin (Hb) and this modification enable Hb to undergo higher rate of auto-oxidation, leading to generation of further intracellular ROS. Objective In this study, we exhibited the possible cause and consequences of Hb modification in cigarette smokers. Methods Twenty-two smokers and 16 nonsmokers, aged 25 to 35 years, having a smoking history of 7–10 years were recruited in this study. Carbonyl content, ferryl form, peroxidase-like and esterase-like activities of Hb were assayed. Free iron release by Hb, erythrocyte membrane-bound Hb and plasma Hb were also measured along with assessment of important biomolecular degradations by Hb. Results and discussion Increase in carbonyl content in Hb indicates its oxidative degradation. Increase in ferryl Hb formation, peroxidase-like activity and decrease in esterase like activity of Hb along with increased release of nonheme iron (from Hb) clearly indicates alteration in physico-chemical properties of Hb in smokers. Moreover, increase in erythrocyte membrane-bound Hb and plasma-free Hb provide further evidences for higher rate of Hb oxidation in smokers’ erythrocyte. The rates of protein, lipid, sugar and DNA degradation were noticed to be higher by smokers’ Hb; and were further attenuated by desferrioxamine as well as mannitol. Conclusion We conclude that in cigarette smokers, there is oxidative degradation of Hb and the degradation causes alteration in its physico-chemical properties, which in turn may degrade different biomolecules in its close vicinity by releasing more iron and production of more superoxide as well as hydroxyl radical.

Temporal-dependent effects of DNA degradation on frozen tissues archived at −80°C

Abstract Frozen tissues, associated with natural history and biological collections, historically have been archived at temperatures between −20°C and −80°C. More recently, the availability of liquid nitrogen systems has enabled the storage of tissue samples (biobanking) at temperatures as low as −196°C. Currently, it is not known how the degree of coldness (e.g., −80°C or −196°C) or longevity (time in storage) impacts preservation of tissue samples. To examine the effects of long-term storage (−80°C and −196°C) on DNA degradation, tissue samples (muscle and liver) archived for 30, 20, 10, or 1 years were obtained from the Natural Science Research Laboratory at Texas Tech University. The integrity of DNA (measured as molecular weight and fragment length) extracted from samples was determined using automated DNA isolation methods followed by microfluidic distribution measurement. DNA distributions were compared using measures of central tendency, a regression-based molecular mass profile, and as a latent variable in a structural equation model. Muscle samples consistently outperformed liver samples in terms of quality of DNA yield. Also, muscle samples exhibited a significant linear relationship with time in which older samples were more degraded than were recent samples. The signal for a temporal effect on DNA was strongest when considering a latent variable of DNA quality based on mode and kurtosis; 37% of the variation in the latent variable was explained by variation in units of time. More recent time points tended to be more similar, but the temporal effect on the latent variable remained strong even when the oldest samples were removed from the analysis. In contrast, integrity of DNA from liver samples did not have a significant linear relationship with time; however, in some years they exhibited non-normally distributed DNA quality metrics that may have reflected sensitivity of liver tissue to degradation during specimen preparation, DNA extraction, or archive parameters. Results indicated that tissue type and temporal effects influenced rates of DNA degradation, with the latter emphasizing the long-term value of biobanking at the coldest temperatures possible (liquid nitrogen storage) to mitigate degradation of biological samples of ever-increasing scientific value.

A Model and Simulation of the Influence of Temperature and Amplicon Length on Environmental DNA Degradation Rates: A Meta-Analysis Approach

Environmental DNA (eDNA) analysis can detect aquatic organisms, including rare and endangered species, in a variety of habitats. Degradation can influence eDNA persistence, impacting eDNA-based species distribution and occurrence results. Previous studies have investigated degradation rates and associated contributing factors. It is important to integrate data from across these studies to better understand and synthesize eDNA degradation in various environments. We complied the eDNA degradation rates and related factors, especially water temperature and amplicon lengths of the measured DNA from 28 studies, and subjected the data to a meta-analysis. In agreement with previous studies, our results suggest that water temperature and amplicon length are significantly related to the eDNA degradation rate. From the 95% quantile model simulation, we predicted the maximum eDNA degradation rate in various combinations of water temperature and amplicon length. Predicting eDNA degradation could be important for evaluating species distribution and inducing innovation (e.g., sampling, extraction, and analysis) of eDNA methods, especially for rare and endangered species with small population size.

High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains

DNA yield varies by anatomical region, and the selection of bone types that yield maximum recovery of DNA is important to maximize the success of human identification of skeletal remains. The goal of our study was to explore inter- and intra-individual variation in DNA content by measuring nuclear DNA quantity and quality and autosomal STR typing success to determine the most promising skeletal elements for bone sampling. To exclude the influence of taphonomic issues as much as possible, three complete male skeletons from a single Second World War mass grave were examined and all representative skeletal element types of the human body were analyzed. Forty-eight different types of bones from the head, torso, arm, leg, hand, and foot were sampled from each skeleton, 144 bones altogether. The samples were cleaned, and half a gram of bone powder was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen). DNA content and rates of DNA degradation were determined with the PowerQuant (Promega), and the Investigator ESSplex SE QS (Qiagen) was used for STR typing. The highest-yielding bones mostly produced the most complete STR profiles. Among the skeletal elements containing on average the most DNA and producing the most complete profiles in all three skeletons examined were metacarpals, metatarsals, and the petrous portion of the temporal bone. Metatarsals and metacarpals can easily be sampled without using a saw, thus reducing potential DNA contamination. Skeletons from the Second World War can be used as a model for poorly preserved skeletal remains, and the results of the investigation can be applied for genetic identification of highly degraded skeletal remains in routine forensic casework. Although the research was limited to only three skeletons found in a unique mass grave, the data obtained could contribute to sampling strategies for identifying old skeletal remains. More Second World War skeletons will be analyzed in the future to investigate inter-bone variation in the preservation of DNA.

Stirring up the relationship between quantified environmental DNA concentrations and exoskeleton‐shedding invertebrate densities

AbstractThe application of eDNA techniques for the detection, monitoring, and conservation of biodiversity holds great promise. While many studies apply eDNA techniques in aquatic systems to determine the presence or absence of a given species, using eDNA for the purpose of species density or biomass predictions remains a challenge, especially for freshwater invertebrates that shed exoskeletons. Here, we aimed to determine whether and how eDNA concentrations relate to exoskeleton‐shedding invertebrate densities. We used microcosms holding different densities of a common invertebrate freshwater species, Daphnia magna. During 2 weeks, we monitored temporal dynamics of eDNA and the eDNA/density relationship by taking water samples and quantifying eDNA concentrations with the droplet digital PCR. The setup included one treatment without and one with homogenization before sampling, to test the effects of admixture on the relation between eDNA concentration and density. Daphnia magna individuals were removed after 1.5 weeks to track DNA degradation rates. In the stagnant water setup, hardly any DNA was detected before D. magna removal. Within days after removal, eDNA concentrations became undetectable. No significant correlation between D. magna density and eDNA concentrations was observed. In the homogenization treatment, a significant positive correlation between eDNA concentration and densities was demonstrated for the days around D. magna removal, albeit with some within‐treatment variability. Our results show that, given adequate time for eDNA production and degradation to stabilize, positive correlations between eDNA and organism densities in water with sufficient homogenization are detectable for exoskeleton‐shedding invertebrates. Therefore, our study indicates that—although difficult—using eDNA to quantify freshwater exoskeleton‐shedding invertebrate densities may be possible under field conditions if circumstances result in frequent homogenization of the water column.

DNA degradation in fish: Practical solutions and guidelines to improve DNA preservation for genomic research.

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.

Comparison of different mathematical models to assess seasonal variations in the longevity of DNA integrity of cooled‐stored stallion sperm

Dynamic assessment of sperm DNA fragmentation (SDF) has shown to give fuller understanding of stallion semen quality; however, there have been limited attempts to use this parameter to investigate seasonal changes in productive functions. The aims of this study were to: (a) establish a reliable mathematical model to describe the longevity of cooled-stored sperm DNA integrity; (b) to examine the effect of seasonal variations on SDF. Ejaculates were cooled to 5°C, and SDF was analysed after 0, 6 and 24hr of storage. The coefficient of determination (R2 ) was calculated after fine-tuning linear (LIN), exponential (EXP) and second order polynomial (POL) models. R2 was significantly higher (p<.001) for POL than for LIN and EXP. The rate of DNA degradation was calculated using the slopes of POL equations. After assessing the rate of change of the POL functions, significant differences between the acceleration of DNA fragmentation were found (p<.01) among seasons, being higher for winter and summer than spring and autumn. In conclusion, DNA analysis of stallion sperm fits better to a second order polynomial mathematical model, being spring the best season to collect and process cooled stallion semen in order to maintain the DNA integrity of the stallion sperm.