Genotyping Success Research Articles

Limited Short-Term Evolution of SARS-CoV-2 RNA-Dependent RNA Polymerase under Remdesivir Exposure in Upper Respiratory Compartments.

The extent of the SARS-CoV-2 short-term evolution under Remdesivir (RDV) exposure and whether it varies across different upper respiratory compartments are not fully understood. Patients hospitalized for COVID-19, with or without RDV therapy, were enrolled and completed up to three visits, in which they provided specimens from four respiratory compartments. Near full-length genome SARS-CoV-2 sequences were obtained from viral RNA, standard lineage and variant assignments were performed, and viral mutations in the RNA-dependent RNA polymerase (RdRp) region-the RDV target gene-were detected and compared between participants with and without RDV, across the four compartments, within participants across visits, and versus a larger sequence dataset. The statistical analysis used a generalized linear mixed-effects model. A total of 139 sequences were obtained from 37 out of the 44 (84%) enrolled participants. The genotyping success varied across respiratory compartments, which ranged from 42% with oropharyngeal specimens to 67% with nasopharyngeal specimens and showed improvement with higher viral loads. No RdRp mutations known to be associated with RDV resistance were identified, and for 34 detected mutations at 32 amino acid positions that are not known as RDV-associated, there was no evidence of any associations with the RDV exposure, respiratory compartment, or time. At least 1 of these 34 mutations were detected in all participants, and some differed from the larger sequence dataset. This study highlighted the SARS-CoV-2 short-term genomic stability within hosts and across upper respiratory compartments, which suggests a lack of evolution of RDV resistance over time. This contributes to our understanding of SARS-CoV-2 genomic dynamics.

Environmental effects on faecal genotyping success in mesocarnivores

AbstractNon-invasive genetic sampling can be used in research, monitoring, and conservation of wild animals to, for example, provide insights into diets, identify individuals and estimate population sizes. Non-invasive genetic sampling may be especially useful to monitor elusive species because DNA can be derived from materials such as hairs and faeces without handling individuals. However, the reliability of the results derived from this technique is dependent on the quality of DNA obtained from samples, which can deteriorate from exposure to environmental conditions and sample age. While freshness of the sample is an important factor, the combined effect of different field conditions on the genotyping success is not fully understood. To address this gap, we systematically investigated the effects of sample age and environmental conditions on genotyping success of faeces, in an experimental setting of four treatments that combined rain and temperature conditions typical of central European climates. We compared the performance of a microsatellite marker set and a SNP panel for red fox (Vulpes vulpes), as well as a microsatellite marker set for pine marten (Martes martes) from faeces resampled over 21 days. We found that genotyping success decreased significantly with sample age, however environmental treatments did not impact the success. Furthermore, the SNPs we used amplified more successfully over time than the respective microsatellites. Therefore, the use of SNPs instead of conventional microsatellites, when using faecal samples for analysis relying on correct amplifications, could be advantageous. We recommend to prioritise the collection of fresh faeces regardless of environmental conditions.

How to find a wolverine: Factors affecting detection at wolverine (Gulo gulo) bait stations in western Canada

AbstractHigh individual detection success enables precise estimates of density and the ability to monitor trends in abundance for wolverine and other low‐density species, information that is critical for the implementation and assessment of conservation measures. We evaluated a dataset that included six different wolverine capture–recapture studies over a large gradient in wolverine (Gulo gulo) density to provide recommendations for increasing detection. We examined factors related to bait station components, habitat, and seasonal timing. Accounting for variation in wolverine density and trap duration, our results suggest that bait stations setups having a run pole, frame, and camera to photograph unique ventral color patterns, in addition to hair snag devices, identify more individual wolverine than those without. The presence of snow is a habitat feature that also increases individual detection. Female detection rates were lower than male detection rates at the onset of the reproductive denning season in late February and early March compared with January and early February. We found latency to detection was independent of wolverine density, but greater in areas with human influence. Relatively high rates of genotyping success (55%) were predicted by even a single guard hair left at bait stations, while underfur required ~15 hairs for similar success. Longer sampling intervals reduced genotyping success in spring, more so for underfur than guard hair. Hair samples acquired from barbwire were of higher quality than those from either alligator clips or gun brushes. To improve individual detection for wolverine inventory and monitoring, we recommend deploying run pole setups in areas with low human disturbance that will retain snow into late spring. Extending the winter trapping effort into April and May could increase the chances of detecting denning females. Latency to detection suggests that traps should be active for more than a month, especially in human‐influenced areas, but genotyping success suggests that traps should also be cleared of hair samples at smaller intervals of a month or less, during late winter/spring.

DNA typing of cyanoacrylate fumed latent fingerprints using GlobalFiler™ and ForenSeq™ Signature Prep kits.

DNA typing of latent fingerprints is highly desirable to increase chances of individualization. We recovered DNA from Cyanoacrylate (CA) fumed fingerprints and used both GlobalFiler™ and ForenSeq™ DNA Signature Prep kits for DNA typing. For GlobalFiler™, samples were processed using a protocol modified for Low Template (LT)-DNA samples (half-volume reactions, 30 cycles) while for ForenSeq™ DNA Signature Prep, samples were processed using a standard protocol and fluorometer-based library quantitation. We evaluated genotyping success and quality of profiles in terms of completeness, Peak Height Ratio/Allele Coverage Ratio, presence of PCR artifacts and drop-in alleles. With GlobalFiler™, average autosomal STR (aSTR) profile completeness was 44.4% with 2-20 pg, 54.3% with 22-60 pg, and 95% with 64-250 pg DNA input. CODIS uploadable profiles were obtained in 2/10, 3/11, and 11/12 samples in these ranges. With ForenSeq™ DNA Signature Prep, average aSTR profile completeness was 19.7% with 1-20 pg and 45.2% with 22-47 pg but increased to 78.3% with 68-122 pg and 86.7% with 618-1000 pg DNA input. Uploadable profiles were obtained in 0/12, 4/11, 4/7, and 3/3 samples for these ranges. Results show very high sensitivity using both kits. Half-volume reactions and 30 cycles had minimal negative effect on Globalfiler™ profile quality, providing support for wider use after validation experiments to routinely improve results from LT samples. A standard protocol for the ForenSeq™ DNA Signature Prep kit was also highly successful with LT DNA obtained from CA-fumed fingerprints with additional information from isometric STR alleles and other markers.

The John Charnley Award: The Impact of Human Leukocyte Antigen Genotype on Bacterial Infection Rates and Successful Eradication in Total Hip Arthroplasty

BackgroundGenetics play an important role in several medical domains; however, the influence of human leukocyte antigen (HLA) genotype on the development of periprosthetic joint infection (PJI) in total hip arthroplasty (THA) remains unknown. The primary aim of this study was to determine if HLA genotype is associated with the development of bacterial PJI in THA. Secondarily, we evaluated the association between HLA genotype and PJI treatment success. MethodsA retrospective, matched, case-control study was performed using prospectively collected data from a single institution. A total of 49 patients who underwent primary THA were included, with a mean follow-up of 8.5 years (range, 4.2 to 12.9). The 23 cases (PJI) and 26 controls (no PJI) were matched for age, sex, follow-up, body mass index, primary diagnosis, and comorbidities (P > .05). High-resolution genetic analysis targeting 11 separate HLA loci was performed in all patients using serum samples. The HLA gene frequencies and carriage rates were determined and compared between cohorts. A subgroup analysis of PJI treatment success (18) and failure (5) was performed. Statistical significance was set at P = .10 for genetic analysis and at 0.05 for all other analyses. ResultsThere were 4 HLA alleles that were significantly associated with the development of PJI. The 3 at-risk alleles included HLA-C∗06:02 (odds ratio 5.25, 95% CI [confidence interval] 0.96 to 28.6, P = .064), HLA-DQA1∗04:01 (P = .096), and HLA-DQB1∗04:02 (P = .096). The single protective allele was HLA-C∗03:04 (odds ratio 0.12, 95% CI 0.01 to 1.10, P = .052). There were no specific HLA alleles that were associated with treatment success or failure. ConclusionsThis study suggests that there are at-risk and protective HLA alleles associated with the development of PJI in THA. To our knowledge, this is the first study to demonstrate an association between patient HLA genotype and the development of PJI. A larger study of the subject matter is necessary and warranted.

Development and validation of a GT-seq panel for genetic monitoring in a threatened species using minimally invasive sampling.

Minimally invasive samples are often the best option for collecting genetic material from species of conservation concern, but they perform poorly in many genomic sequencing methods due to their tendency to yield low DNA quality and quantity. Genotyping-in-thousands by sequencing (GT-seq) is a powerful amplicon sequencing method that can genotype large numbers of variable-quality samples at a standardized set of single nucleotide polymorphism (SNP) loci. Here, we develop, optimize, and validate a GT-seq panel for the federally threatened northern Idaho ground squirrel (Urocitellus brunneus) to provide a standardized approach for future genetic monitoring and assessment of recovery goals using minimally invasive samples. The optimized panel consists of 224 neutral and 81 putatively adaptive SNPs. DNA collected from buccal swabs from 2016 to 2020 had 73% genotyping success, while samples collected from hair from 2002 to 2006 had little to no DNA remaining and did not genotype successfully. We evaluated our GT-seq panel by measuring genotype discordance rates compared to RADseq and whole-genome sequencing. GT-seq and other sequencing methods had similar population diversity and F ST estimates, but GT-seq consistently called more heterozygotes than expected, resulting in negative F IS values at the population level. Genetic ancestry assignment was consistent when estimated with different sequencing methods and numbers of loci. Our GT-seq panel is an effective and efficient genotyping tool that will aid in the monitoring and recovery of this threatened species, and our results provide insights for applying GT-seq for minimally invasive DNA sampling techniques in other rare animals.

Efficient species identification for Pacific salmon genetic monitoring programs.

Genetic monitoring of Pacific salmon in the Columbia River basin provides crucial information to fisheries managers that is otherwise challenging to obtain using traditional methods. Monitoring programs such as genetic stock identification (GSI) and parentage-based tagging (PBT) involve genotyping tens of thousands of individuals annually. Although rare, these large sample collections inevitably include misidentified species, which exhibit low genotyping success on species-specific Genotyping-in-Thousands by sequencing (GT-seq) panels. For laboratories involved in large-scale genotyping efforts, diagnosing non-target species and reassigning them to the appropriate monitoring program can be costly and time-consuming. To address this problem, we identified 19 primer pairs that exhibit consistent cross-species amplification among salmonids and contain 51 species informative variants. These genetic markers reliably discriminate among 11 salmonid species and two subspecies of Cutthroat Trout and have been included in species-specific GT-seq panels for Chinook Salmon, Coho Salmon, Sockeye Salmon, and Rainbow Trout commonly used for Pacific salmon genetic monitoring. The majority of species-informative amplicons (16) were newly identified from the four existing GT-seq panels, thus demonstrating a low-cost approach to species identification when using targeted sequencing methods. A species-calling script was developed that is tailored for routine GT-seq genotyping pipelines and automates the identification of non-target species. Following extensive testing with empirical and simulated data, we demonstrated that the genetic markers and accompanying script accurately identified species and are robust to missing genotypic data and low-frequency, shared polymorphisms among species. Finally, we used these tools to identify Coho Salmon incidentally caught in the Columbia River Chinook Salmon sport fishery and used PBT to determine their hatchery of origin. These molecular and computing resources provide a valuable tool for Pacific salmon conservation in the Columbia River basin and demonstrate a cost-effective approach to species identification for genetic monitoring programs.

A simple and effective method to enrich endogenous DNA from mammalian faeces.

Utilization of faeces has long been a popular approach for genetic and ecological studies of wildlife. However, the success of molecular marker genotyping and genome resequencing is often unpredictable due to insufficient enrichment of endogenous DNA in the total faecal DNA that is dominated by bacterial DNA. Here, we report a simple and cheap method named PEERS to predominantly lyse animal cells over bacteria by using sodium dodecyl sulphate so as to discharge endogenous DNA into liquid phase before bacterial DNA. By brief centrifugation, total DNA with enriched endogenous fraction can be extracted from the supernatant using routine methods. Our assessments showed that the endogenous DNA extracted by PEERS was significantly enriched for various types of faeces from different species, preservation time and conditions. It significantly improves the genotyping correctness and efficiency of genome resequencing with the total additional cost of $ 0.1 and a short incubation step to treat a faecal sample. We also provide methods to assess the enrichment efficiency of mitochondrial and nuclear DNA and models to predict the usability of faecal DNA for genotyping of short tandem repeat, single-nucleotide polymorphism and whole-genome resequencing.

Quantitative color analysis of burned bone to predict DNA quantity, quality, and genotyping success.

Badly burned skeletal remains are commonly submitted to forensic laboratories for victim identification via DNA analysis methods. Burned skeletal remains present many challenges for DNA analysis as they can contain low amounts of DNA which can also be damaged and degraded, resulting in partial or no STR profiles. Therefore, a simple, but effective screening method that identifies which samples may provide the most successful STR or mtDNA typing results for identification would enable forensic laboratories to save time, money, and resources. One metric that can be used and a screening method is the color of burned bone, as bone color changes with exposure to fire as temperature and length of exposure increase. This research developed a quantitative screening method based on the surface color of burned bone. The different visual bone colors (light brown, dark brown, black, gray, and white) were quantified using the Commission on Illumination L*a*b color space. These values were then compared to DNA yield, STR, and mtDNA profile completeness to identify whether the L*a*b values can predict genotyping success. A Bayesian network was constructed to determine the probability of STR typing success, given a set of L*a*b values. Results demonstrated that samples with an a* value greater than or equal to one and b* value greater than eight (light brown and dark brown burned samples) were the most predictive of STR typing success and mtDNA typing success. A decision tree for processing burned bones was constructed based on the color value thresholds.

Evaluating genotyping-in-thousands by sequencing as a geneticmonitoring tool for a climate sentinel mammal using non-invasive and archival samples.

Genetic tools for wildlife monitoring can provide valuable information on spatiotemporal population trends and connectivity, particularly in systems experiencing rapid environmental change. Multiplexed targeted amplicon sequencing techniques, such as genotyping-in-thousands by sequencing (GT-seq), can provide cost-effective approaches for collecting genetic data from low-quality and quantity DNA samples, making them potentially useful for long-term wildlife monitoring using non-invasive and archival samples. Here, we developed a GT-seq panel as a potential monitoring tool for the American pika (Ochotona princeps) and evaluated its performance when applied to traditional, non-invasive, and archival samples, respectively. Specifically, we optimized a GT-seq panel (307 single nucleotide polymorphisms (SNPs)) that included neutral, sex-associated, and putatively adaptive SNPs using contemporary tissue samples (n = 77) from the Northern Rocky Mountains lineage of American pikas. The panel demonstrated high genotyping success (94.7%), low genotyping error (0.001%), and excellent performance identifying individuals, sex, relatedness, and population structure. We subsequently applied the GT-seq panel to archival tissue (n = 17) and contemporary fecal pellet samples (n = 129) collected within the Canadian Rocky Mountains to evaluate its effectiveness. Although the panel demonstrated high efficacy with archival tissue samples (90.5% genotyping success, 0.0% genotyping error), this was not the case for the fecal pellet samples (79.7% genotyping success, 28.4% genotyping error) likely due to the exceptionally low quality/quantity of recovered DNA using the approaches implemented. Overall, our study reinforced GT-seq as an effective tool using contemporary and archival tissue samples, providing future opportunities for temporal applications using historical specimens. Our results further highlight the need for additional optimization of sample and genetic data collection techniques prior to broader-scale implementation of a non-invasive genetic monitoring tool for American pikas.

Individual genotypes from environmental DNA: Fingerprinting snow tracks of three large carnivore species.

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies.

Capturing environmental DNA in snow tracks of polar bear, Eurasian lynx and snow leopard towards individual identification

Polar bears (Ursus maritimus), Eurasian lynx (Lynx lynx) and snow leopards (Panthera uncia) are elusive large carnivores inhabiting snow-covered and remote areas. Their effective conservation and management are challenged by inadequate population information, necessitating development of novel data collection methods. Environmental DNA (eDNA) from snow tracks (footprints in snow) has identified species based on mitochondrial DNA, yet its utility for individual-based analyses remains unsolved due to challenges accessing the nuclear genome. We present a protocol for capturing nuclear eDNA from polar bear, Eurasian lynx and snow leopard snow tracks and verify it through genotyping at a selection of microsatellite markers. We successfully retrieved nuclear eDNA from 87.5% (21/24) of wild polar bear snow tracks, 59.1% (26/44) of wild Eurasian lynx snow tracks, and the single snow leopard sampled. We genotyped over half of all wild polar bear samples (54.2%, 13/24) at five loci, and 11% (9/44) of wild lynx samples and the snow leopard at three loci. Genotyping success from Eurasian lynx snow tracks increased to 24% when tracks were collected by trained rather than untrained personnel. Thirteen wild polar bear samples comprised 11 unique genotypes and two identical genotypes; likely representing 12 individual bears, one of which was sampled twice. Snow tracks show promise for use alongside other non-invasive and conventional methods as a reliable source of nuclear DNA for genetic mark-recapture of elusive and threatened mammals. The detailed protocol we present has utility for broadening end user groups and engaging Indigenous and local communities in species monitoring.

Development of a SNaPshot assay for the genotyping of organellar SNPs in four closely related pines

Mitochondrial (mtDNA) and chloroplast (cpDNA) polymorphisms are valuable resources to study past demographic changes, phylogenetics and evolution, especially in forest tree species, where these genomes are haploid and uniparentally transferred. The organellar markers were usually scored separately using direct sequencing or PCR-based approaches, which can be time-consuming and expensive, especially in large-scale population genetics research. In this study, we developed an efficient and cost-effective SNaPshot assay for genotyping preselected ­mtDNA and cpDNA polymorphism in four closely related pine species including Scots pine (Pinus sylvestris L.) and three taxa from the Pinus mugo complex. We validated the method by genotyping the samples derived from 12 populations of the species from their wide geographical distribution range in Europe. The results proved high accuracy of the method with a genotyping success rate of 99.7%. The set of assayed markers shows significant genetic variation. By using multiplex SNaPshot assay, we provided an efficient and sensitive molecular tool for intra- and interspecific genetic analyses. The presented protocol is useful for fast and relatively cheap SNP genotyping of organelle genome of closely related pine species. The assayed SNPs allow studying the species discrimination and detailed investigations of their population history and structure. Given its numerous benefits and efficient genotyping rate, the SNaPshot method appears to be a valuable and practical resource for studying the genetic makeup of forest tree species. Particularly, it proves to be advantageous for population genetics.

Self-compatibility in yeast is selected for reproductive assurance not population-level compatibility.

In haploid species, sexual reproduction by selfing lacks the common benefits from recombination and is indistinguishable from asexual reproduction at the genetic level. Nevertheless, the evolution of self-compatibility, known as homothallism in organisms with mating types, has occurred hundreds of times in fungi. Two main hypotheses have been proposed for the evolution of homothallism. First, that homothallism offers reproductive assurance, which is especially important when species have an obligatory sexual phase in their lifecycle. Second, that homothallism is associated with population-level compatibility, increasing the chance of outbreeding. Here, we test these hypotheses using the fission yeast Schizosaccharomyces pombe, which is homothallic by mating-type switching, leveraging natural variation for switching efficiency in this species. Combining empirical tests with cellular automaton simulations, we show that homothallism by switching increases mating success of switching genotypes, but does not affect population-level compatibility. Experiments show that outcrossing is actually reduced under homothallism. This reduction in outcrossing is explained by our simulations, which show that due to local mating, gametes that mated through intraclonal selfing are no longer available for outcrossing. Our results suggest that the recurrent evolution of haploid self-compatibility is likely driven by selection for mating assurance, not to increase the potential for outcrossing.

No evidence for schistosome parasite fitness trade-offs in the intermediate and definitive host

BackgroundThe trematode parasite Schistosoma mansoni uses an aquatic snail intermediate and a vertebrate definitive host to complete its life cycle. We previously showed that a key transmission trait—the number of cercariae larvae shed from infected Biomphalaria spp. snails—varies significantly within and between different parasite populations and is genetically controlled by five loci. We investigated the hypothesis that the success of parasite genotypes showing high propagative fitness in the intermediate snail host may be offset by lower reproductive fitness in the definitive vertebrate host.MethodsWe investigated this trade-off hypothesis by selecting parasite progeny producing high or low number of larvae in the snail and then comparing fitness parameters and virulence in the rodent host. We infected inbred BALB/c mice using two Schistosoma mansoni parasite lines [high shedder (HS) and low shedder (LS) lines] isolated from F2 progeny generated by genetic crosses between SmLE (HS parent) and SmBRE (LS parent) parasites. We used the F3 progeny to infect two populations of inbred Biomphalaria glabrata snails. We then compared life history traits and virulence of these two selected parasite lines in the rodent host to understand pleiotropic effects of genes determining cercarial shedding in parasites infecting the definitive host.ResultsHS parasites shed high numbers of cercariae, which had a detrimental impact on snail physiology (measured by laccase-like activity and hemoglobin rate), regardless of the snail genetic background. In contrast, selected LS parasites shed fewer cercariae and had a lower impact on snail physiology. Similarly, HS worms have a higher reproductive fitness and produced more viable F3 miracidia larvae than LS parasites. This increase in transmission is correlated with an increase in virulence toward the rodent host, characterized by stronger hepato-splenomegaly and hepatic fibrosis.ConclusionsThese experiments revealed that schistosome parasite propagative and reproductive fitness was positively correlated in intermediate and definitive host (positive pleiotropy). Therefore, we rejected our trade-off hypothesis. We also showed that our selected schistosome lines exhibited low and high shedding phenotype regardless of the intermediate snail host genetic background. ​Graphical

A Bayesian approach to incorporate structural data into the mapping of genotype to antigenic phenotype of influenza A(H3N2) viruses.

Surface antigens of pathogens are commonly targeted by vaccine-elicited antibodies but antigenic variability, notably in RNA viruses such as influenza, HIV and SARS-CoV-2, pose challenges for control by vaccination. For example, influenza A(H3N2) entered the human population in 1968 causing a pandemic and has since been monitored, along with other seasonal influenza viruses, for the emergence of antigenic drift variants through intensive global surveillance and laboratory characterisation. Statistical models of the relationship between genetic differences among viruses and their antigenic similarity provide useful information to inform vaccine development, though accurate identification of causative mutations is complicated by highly correlated genetic signals that arise due to the evolutionary process. Here, using a sparse hierarchical Bayesian analogue of an experimentally validated model for integrating genetic and antigenic data, we identify the genetic changes in influenza A(H3N2) virus that underpin antigenic drift. We show that incorporating protein structural data into variable selection helps resolve ambiguities arising due to correlated signals, with the proportion of variables representing haemagglutinin positions decisively included, or excluded, increased from 59.8% to 72.4%. The accuracy of variable selection judged by proximity to experimentally determined antigenic sites was improved simultaneously. Structure-guided variable selection thus improves confidence in the identification of genetic explanations of antigenic variation and we also show that prioritising the identification of causative mutations is not detrimental to the predictive capability of the analysis. Indeed, incorporating structural information into variable selection resulted in a model that could more accurately predict antigenic assay titres for phenotypically-uncharacterised virus from genetic sequence. Combined, these analyses have the potential to inform choices of reference viruses, the targeting of laboratory assays, and predictions of the evolutionary success of different genotypes, and can therefore be used to inform vaccine selection processes.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

Evaluating predator control using two non-invasive population metrics: a camera trap activity index and density estimation from scat genotyping

Context Invasive mammalian predators are a threat to biodiversity and agriculture globally, yet management outcomes for lethal predator control remain difficult to monitor and evaluate. Understanding whether changes in activity indices correspond to true changes in population density will help inform effective monitoring and management programs. Aims The aim of this study was to evaluate the effect of poison baiting on invasive red fox (Vulpes vulpes) populations using two alternative population metrics: fox activity from camera trap surveys and density estimation from scat genetic analysis. Methods We conducted before–after control–impact studies in two regions of semi-arid Australia (Wimmera and Mallee) by monitoring paired non-treatment and treatment sites during unbaited and baited periods. We estimated the effects of poison baiting on: (1) a monthly fox activity index, derived from an array of 10 off-road camera traps per site; and (2) fox density. To estimate density, we collected fox scats along 14-km transects, identified individuals using polymorphic microsatellite DNA markers and fitted spatially explicit capture–recapture models. Key results Fox activity remained consistently low at all sites except the Mallee non-treatment. The top-ranked models of fox activity and density contained an interaction between treatment and period, with an interactive and additive effect of region, respectively. However, there was little evidence that baiting reduced fox activity or density. In the unbaited period, fox densities ranged from 0.69 (95% CI: 0.47–1.0) to 1.06 (95% CI: 0.74–1.51) foxes km−2 and were similar across regions. Conclusions Camera traps have the potential to provide continuous index-based measures of fox populations but may not record sufficient observations to detect change. Indices can also be confounded by variations in animal behaviour. Scat genetic analysis is a viable option for providing direct estimates of population change at specific snapshots in time; however, this approach is considerably more expensive, and large sample sizes may be required if genotyping success is low. Implications Our study presents a rare example of multiple concurrent – and non-invasive – monitoring techniques to evaluate the effectiveness of predator control. We highlight the value of rigorous study designs and high-quality density information for designing predator management and monitoring programs.

HIV-1C in-House RNA-Based Genotyping Assay for Detection of Drug Resistance Mutations in Samples with Low-Level Viral Loads.

Monitoring HIV-1 drug resistance mutations (DRM) in treated patients on combination antiretroviral therapy (cART) with a detectable HIV-1 viral load (VL) is important for the selection of appropriate cART. Currently, there is limited data on HIV DRM at low-level viremia (LLV) (VL 401-999 copies/mL) due to the use of a threshold of VL ≥1000 copies/mL for HIV DRM testing. We here assess the performance of an in-house HIV drug resistance genotyping assay using plasma for the detection of DRM at LLV. We used a total of 96 HIV plasma samples from the population-based Botswana Combination Prevention Project (BCPP). The samples were stratified by VL groups: 50 samples had LLV, defined as 401-999 copies/mL, and 46 had ≥1000 copies/mL. HIV pol (PR and RT) region was amplified and sequenced using an in-house genotyping assay with BigDye sequencing chemistry. Known HIV DRMs were identified using the Stanford HIV Drug Resistance Database. Genotyping success rate between the two groups was estimated and compared using the comparison of proportions test. The overall genotyping success rate was 79% (76/96). For VL groups, the genotyping success was 72% (36/50) at LLV and 87% (40/46) at VL ≥1000 copies/mL. Among generated sequences, the overall prevalence of individuals with at least 1 major or intermediate-associated DRM was 24% (18/76). The proportions of NNRTI-, NRTI- and PI-associated resistance mutations were 28%, 24%, and 0%, respectively. The most predominant mutations detected were K103N (18%) and M184V (12%) in NNRTI- and NRTI-associated mutations, respectively. The prevalence of DRM was 17% (6/36) at LLV and 30% (12/40) at VL ≥1000 copies/mL. The in-house HIV genotyping assay successfully genotyped 72% of LLV samples and was able to detect 17% of DRM amongst them. Our results highlight the possibility and clinical significance of genotyping HIV among individuals with LLV.

Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.