Non-invasive DNA Sampling Research Articles

Utility of liquid biopsy for predicting cancer type and informing treatment of carcinoma with unknown primary.

e13606 Background: The advent of next-generation sequencing has greatly facilitated physicians in recommending tailored treatments for cancer patients. Despite these advancements, the dependence on the primary tumor site remains crucial for accurate diagnoses, posing a substantial obstacle in cases of carcinomas of unknown primary (CUPs) and hindering optimal patient care. To address this challenge, we propose the implementation of a machine learning model capable of identifying the tissue of origin (TOO) across 16 cancer types. Current standards gravitate to the use of tumor tissue for identifying primary tumor site. Leveraging the advantages of cell-free DNA (cfDNA), our approach provides a non-invasive and highly sensitive alternative to traditional biopsy or radiography methods. Methods: Cell-free DNA was extracted from patients diagnosed with carcinoma of known origin. The model underwent training using a dataset comprising 2,793 cfDNA samples, while the validation set consisted of 1,863 samples. The training and validation cohorts exhibited comparable demographics in terms of sex, age, and cancer types. Employing low-coverage whole genome sequencing (WGS), samples were analyzed to capture cfDNA features, including DNA fragment size distribution, copy number variation, nucleosome coverage pattern, mutational signature, and hotspot regions associated with cancer-related mutations. The final prediction model's accuracy was evaluated based on top 1 and top 2 predictions in accordance with the true tumor site of origin. Results: Overall accuracy of TOO predictions in the training cohort is 80.20% (2240/2793) for top 1 site and 90.26% (2521/2793) for top 2 sites. Overall accuracy of TOO predictions in the validation cohort is 81.80% (1524/1863) for top 1 site and 90.07% (1678/1863) for top 2 sites. Accuracy of tumor site prediction in the validation cohort is highest for lung cancer (Top 1 = 95.9%; Top 2 = 98.0%) tumor sites and lowest for biliary system and kidney tumor types (Top 1 = 54.3% & 57.4%; Top 2 = 71.6% & 63.2%). In the clinical setting, TOO estimation of 12/16 patients with CUP were identified to be in concordance with majority pathological consensus. Of note, two patients with CUP were diagnosed as lung cancer patients through TOO estimation. Molecular biomarkers of lung adenocarcinoma were also identified through targeted sequencing which corroborated the diagnosis, and chemoradiotherapy was recommended to the patient. Conclusions: We developed an accurate predictor of TOO by leveraging machine learning algorithm and incorporating cfDNA mutational, fragmentomic, and epigenetic features. Our results demonstrate high concordance between model-predicted and actual tumor sites across all examined cancer types. This model shows promise in offering valuable insights for the identification of cancers with an unknown primary origin using non-invasive cell free DNA samples.

Addressing Misidentification in Noninvasive DNA Sampling Using Bayesian Approach and Simulations

Addressing Misidentification in Noninvasive DNA Sampling Using Bayesian Approach and Simulations

Assessing the recovery of Y chromosome microsatellites with population genomic data using Papio and Theropithecus genomes

Y chromosome markers can shed light on male-specific population dynamics but for many species no such markers have been discovered and are available yet, despite the potential for recovering Y-linked loci from available genome sequences. Here, we investigated how effective available bioinformatic tools are in recovering informative Y chromosome microsatellites from whole genome sequence data. In order to do so, we initially explored a large dataset of whole genome sequences comprising individuals at various coverages belonging to different species of baboons (genus: Papio) using Y chromosome references belonging to the same genus and more distantly related species (Macaca mulatta). We then further tested this approach by recovering Y-STRs from available Theropithecus gelada genomes using Papio and Macaca Y chromosome as reference sequences. Identified loci were validated in silico by a) comparing within-species relationships of Y chromosome lineages and b) genotyping male individuals in available pedigrees. Each STR was selected not to extend in its variable region beyond 100 base pairs, so that loci can be developed for PCR-based genotyping of non-invasive DNA samples. In addition to assembling a first set of Papio and Theropithecus Y-specific microsatellite markers, we released TYpeSTeR, an easy-to-use script to identify and genotype Y chromosome STRs using population genomic data which can be modulated according to available male reference genomes and genomic data, making it widely applicable across taxa.

Empirical Data Suggest That the Kashmir Musk Deer (Moschus cupreus, Grubb 1982) Is the One Musk Deer Distributed in the Western Himalayas: An Integration of Ecology, Genetics and Geospatial Modelling Approaches.

Insufficient research has been conducted on musk deer species across their distribution range, primarily because of their elusive behaviour and the fact they occupy remote high-altitude habitats in the Himalayas above 2500 m. The available distribution records, primarily derived from ecological studies with limited photographic and indirect evidence, fail to provide comprehensive information on the species distribution. Consequently, uncertainties arise when attempting to determine the presence of specific taxonomic units of musk deer in the Western Himalayas. This lack of knowledge hampers species-oriented conservation efforts, as there need to be more species-specific initiatives focused on monitoring, protecting, and combatting the illegal poaching of musk deer for their valuable musk pods. We used transect surveys (220 trails), camera traps (255 cameras), non-invasive DNA sampling (40 samples), and geospatial modelling (279 occurrence records) to resolve the taxonomic ambiguity, and identify the suitable habitat of musk deer (Moschus spp.) in Uttarkashi District of Uttarakhand and the Lahaul-Pangi landscape of Himachal Pradesh. All the captured images and DNA-based identification results confirmed the presence of only Kashmir musk deer (KDM) (Moschus cupreus) in Uttarakhand and Himachal Pradesh. The results suggest that KMD inhabit a narrow range of suitable habitats (6.9%) of the entire Western Himalayas. Since all evidence indicates that only KMD are present in the Western Himalayas, we suggest that the presence of other species of musk deer (Alpine musk deer and Himalayan musk deer) was wrongly reported. Therefore, future conservation plans and management strategies must focus only on KMD in the Western Himalayas.

Unexpected and spatially structured genetic diversity of the relict population of the endangered corsican land snail Tyrrhenaria ceratina

Population genetics of threatened species provides information about evolutionary pressures over those populations and thus may inform conservation management strategies. However, conservation genetics still has a low impact on conservation practices. This study’s aim is to integrate genetics in the conservation management of the only-known population of an extremely narrow-range endemic Corsican snail – Tyrrhenaria ceratina –, whose distribution area is restricted to the Ricantu site in Corsica. Using non-invasive DNA samples of 210 individuals, we amplified 13 microsatellites loci to assess the population viability, genetic structure and demographic history of the population, along with the estimation of the historical and contemporary gene flow between identified genetic clusters. We also estimated the dispersal ability of the species. Our results showed a surprisingly high genetic diversity, along with a pattern of isolation by distance (IBD) and a strongly spatialized genetic structure. Furthermore, we underlined a low functional connectivity, along with evidence of a recent decline in the population size, which are both likely due to a historical fragmentation between the sampled areas, caused by anthropization. Overall, this study allows to provide a first insight about the functioning of the population, to guide future conservation actions for the species.

Comparing minimum number of individuals and abundance from non-invasive DNA sampling and camera trapping in the red fox (Vulpes vulpes)

Applying the most appropriate sampling method is essential for estimating population size. Sampling methods and techniques to estimate abundance may be limited by environmental characteristics, species traits, specific requirements of the techniques, or the economic resources to carry out the sampling. Thus, evaluating multiple sampling methods in monitoring populations is essential for establishing effective conservation strategies. In this study, we compare two of the most commonly used sampling methods with the red fox (Vulpes vulpes) as the type species. On the one hand, we compared the minimum number of individuals (NI) detected by camera trapping, identifying individuals by morphological characteristics with the minimum number of individuals detected by DNA faeces and a set of 16 microsatellites. On the other hand, we estimated abundance by performing an N-mixture model using information from camera-traps to study the relationship between abundance and the minimum number of individuals detected. Results showed that the minimum NI provided by camera trapping was slightly higher than that of DNA faecal genotyping, with 23.66 and 19 individuals, respectively. In addition, abundance and NI detected by camera trapping showed a positive relationship. In contrast, there was a non-significant negative relationship between NI detected by faecal DNA and abundance estimates. Our results suggest using the minimum number of photo-identified individuals as a reliable index to study variation in red fox abundance when other advanced methods cannot be implemented in the study of population size. However, it is necessary to improve the methods of faecal sampling to study the relationship with camera-trap data.

Spatial variation in red deer density in a transboundary forest ecosystem

Forests in Europe are exposed to increasingly frequent and severe disturbances. The resulting changes in the structure and composition of forests can have profound consequences for the wildlife inhabiting them. Moreover, wildlife populations in Europe are often subjected to differential management regimes as they regularly extend across multiple national and administrative borders. The red deer Cervus elaphus population in the Bohemian Forest Ecosystem, straddling the Czech-German border, has experienced forest disturbances, primarily caused by windfalls and bark beetle Ips typographus outbreaks during the past decades. To adapt local management strategies to the changing environmental conditions and to coordinate them across the international border, reliable estimates of red deer density and abundance are highly sought-after by policymakers, wildlife managers, and stakeholders. Covering a 1081-km2 study area, we conducted a transnational non-invasive DNA sampling study in 2018 that yielded 1578 genotyped DNA samples from 1120 individual red deer. Using spatial capture-recapture models, we estimated total and jurisdiction-specific abundance of red deer throughout the ecosystem and quantified the role of forest disturbance and differential management strategies in shaping spatial heterogeneity in red deer density. We hypothesised that (a) forest disturbances provide favourable habitat conditions (e.g., forage and cover), and (b) contrasting red deer management regimes in different jurisdictions create a differential risk landscape, ultimately shaping density distributions. Overall, we estimated that 2851 red deer (95% Credible Interval = 2609–3119) resided in the study area during the sampling period, with a relatively even overall sex ratio (1406 females, 95% CI = 1229–1612 and 1445 males, 95% CI = 1288–1626). The average red deer density was higher in Czechia (3.5 km−2, 95% CI = 1.2–12.3) compared to Germany (2 km−2, 95% CI = 0.2–11). The effect of forest disturbances on red deer density was context-dependent. Forest disturbances had a positive effect on red deer density at higher elevations and a negative effect at lower elevations, which could be explained by partial migration and its drivers in this population. Density of red deer was generally higher in management units where hunting is prohibited. In addition, we found that sex ratios differed between administrative units and were more balanced in the non-intervention zones. Our results show that the effect of forest disturbances on wild ungulates is modulated by additional factors, such as elevation and ungulate management practices. Overall density patterns and sex ratios suggested strong gradients in density between administrative units. With climate change increasing the severity and frequency of forest disturbances, population-level monitoring and management are becoming increasingly important, especially for wide-ranging species as both wildlife and global change transcend administrative boundaries.

Combining methods for non-invasive fecal DNA enables whole genome and metagenomic analyses in wildlife biology.

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, or dangerous species. Integrating genomic techniques that use non-invasive biological sampling with advances in computational approaches can benefit and inform wildlife conservation and management. Here, we used non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at six X-fold coverage) and metagenomic sequences, combining widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated was demonstrated through genome scale and metagenomic analyses of zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses identified taxa that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of this approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular and bioinformatic analyses presented here contributes towards the expansion and application of genomic techniques to conservation science and practice.

Scat DNA as a non-invasive method for estimating the abundance of the vulnerable mala (Lagorchestes hirsutus)

Context Population-monitoring programs often use direct (e.g. live capture or spotlighting) or indirect (e.g. scats sightings) observations to estimate population abundance. Such methods, however, are often inadequate for rare, elusive, or cryptic species due to the difficulty in achieving sufficient encounters or detection rates. The mala (Lagorchestes hirsutus), a small native Australian macropod, listed as Vulnerable by the IUCN, is difficult to capture, susceptible to capture myopathy, and not easily sighted in their dense habitat; consequently, the population size cannot always be estimated. The use of molecular markers to identify individual genotypes from non-invasively collected samples is increasingly being used in wildlife conservation and may be an alternative approach for mala. Aim The aim of this study was to evaluate the efficacy of non-invasive scat DNA sampling to estimate the population abundance of mala. Methods A panel of microsatellite markers was developed for the identification of individual mala via profiling of their scats. Scats were systematically collected from a wild mala population located in an 1100-ha fenced reserve in Western Australia. Individual genotypes were determined using the microsatellite markers, and the abundance of mala was estimated using the genotypes with spatially explicit capture–recapture (SECR) and mark–resight analyses. Key results The genetic markers proved variable and with sufficient exclusionary power to confidently identify unique individuals (mean locus genotyping error rate: 3.1%). Individual genetic identification from scat sampling, when used with traditional mark–recapture/resight analytical models, provides feasible estimates of population abundance. This is the first reliable abundance estimate of this mala population, suggesting a >70% increase in population size since the initial reintroduction of 64 individuals in 2011–13. Conclusions Given the inherent difficulties in surveying mala, this approach would be valuable to ensure effective monitoring of the few remaining fenced and island mala populations to prevent further decline of this vulnerable species. Implications This is the first study to identify species-specific microsatellite markers for mala and use genetic-capture sampling with scat DNA to estimate the abundance of a mala population. The study provides an evaluation of a valuable species monitoring technique that can be applied to other rare, elusive, or cryptic threatened species.

Current Genetic Structure Analysis of Leopard Cats Reveals a Weak Disparity Trend in Subpopulations in Beijing, China.

Simple SummaryHabitat fragmentation is an important factor leading to the decline in the leopard cat population in Beijing. Habitat loss may further result in population shrinkage, which increases the risk of inbreeding and genetic decline. To reveal the segregation effects of highway construction and infrastructure expansion on population genetic variation, this study analyzed the genetic structure of leopard cats in five nature reserves in the mountain surroundings of Beijing. The results showed that a mild disparity trend exists in Baihuashan and Songshan subpopulations, due to habitat segregation and dispersal difficulties. We suggest that the genetic structures of the leopard cat population be monitored every 5 years to detect any changes. If needed, individuals can be artificially exchanged among different subpopulations to maintain the viability of this wild cat in Beijing. In the face of habitat shrinkage and segregation, the survival of wild cats looks bleak. Interpreting their population genetic structure during habitat fragmentation is critical in planning effective management strategies. To reveal the segregation effects of road construction and human settlements on the population genetic structure, we analyzed non-invasive fecal DNA samples from leopard cats (Prionailurus bengalensis) from five nature reserves in mountainous areas around Beijing. We focused on microsatellite markers. A total of 112 individual leopard cats were identified among 601 samples of scat, and moderate population genetic diversity was detected. Microsatellite-marker-based genetic differentiation (Fst) and gene flow (Nm) showed a weak trend toward discrepancies in the Baihuashan and Songshan subpopulations, which indicated habitat fragmentation effects on individual dispersal. Because the segregated subpopulations may suffer a high risk of genetic diversity loss, we suggest that their genetic structure be monitored with more molecular markers to detect any changes, and that female individuals be artificially introduced as needed to maintain the viability of the leopard cat subpopulations in Beijing.

Corrigendum: Blood, sweat and tears: a review of non-invasive DNA sampling

Corrigendum: Blood, sweat and tears: a review of non-invasive DNA sampling

Wolf monitoring in Scandinavia: evaluating counts of packs and reproduction events

AbstractLarge carnivores are elusive and use large areas, which causes monitoring to be challenging and costly. Moreover, management to reduce conflicts and simultaneously ensure long‐term population viability require precise population estimates. In Scandinavia, the monitoring of wolves (Canis lupus) is primarily based on counting packs, identifying reproduction, and genetically identifying territorial wolves from noninvasive DNA samples. We assessed the reliability of wolf monitoring in Scandinavia by estimating the detectability of territorial pairs, packs, and reproduction. Our data, comprising snow‐tracking data and DNA‐identified individuals from 2005–2016, covered 11 consecutive winter monitoring seasons (Oct–Mar). Among 343 cases where we identified a wolf pack, territorial wolves were also detected in the same area during the previous season in 323 (94.2%) cases. In only 6 of the remaining 20 cases, there was no prior knowledge of territorial wolves in the area. Among the 328 detected reproduction events (litter born to a pack), we detected 97% during the monitoring period and identified the rest ≥1 year later from kinship assessments of all DNA‐detected individuals. These results suggest that we failed to detect only few packs with reproduction events during the monitoring season that followed breeding. Yearly monitoring of territorial individuals and continuous updates of the pedigree allowed us to retrospectively identify reproduction events and packs that were not identified earlier.

Blood, sweat and tears: a review of non-invasive DNA sampling

Blood, sweat and tears: a review of non-invasive DNA sampling

The Cantabrian capercaillie: A population on the edge

The capercaillie Tetrao urogallus - the world's largest grouse- is a circumboreal forest species, which only two remaining populations in Spain: one in the Cantabrian mountains in the west and the other in the Pyrenees further east. Both have shown severe declines, especially in the Cantabrian population, which has recently been classified as “Critically Endangered”. To develop management plans, information on demographic parameters is necessary to understand and forecast population dynamics. We used spatial capture-recapture (SCR) modeling and non-invasive DNA samples to estimate the current population size in the whole Cantabrian mountain range. In addition, for the assessment of population status, we analyzed the population trajectory over the last 42 years (1978–2019) at 196 leks on the Southern slope of the range, using an integrated population model with a Dail-Madsen model at its core, combined with a multistate capture-recapture model for survival and a Poisson regression for productivity. For 2019, we estimate the size of the entire population at 191 individuals (95% BCI 165–222) for an estimated 60 (48–78) females and 131 (109–157) males. Since the 1970s, our study estimates a shrinkage of the population range by 83%. The population at the studied leks in 2019 was at about 10% of the size estimated for 1978. Apparent annual survival was estimated at 0.707 (0.677–0.735), and per-capita recruitment at 0.233 (0.207–0.262), and insufficient to maintain a stable population. We suggest work to improve the recruitment (and survival) and manage these mountain forests for capercaillie conservation. Also, in the future, management should assess the genetic viability of this population.

Estimating dispersal using close kin dyads: The kindisperse R package

Investigating dispersal in animal populations can be difficult, particularly for taxa that are hard to directly observe such as those that are small or rare. A promising solution may come from new approaches that use genome-wide sequence data to detect close kin dyads and estimate dispersal parameters from the distribution of these dyads. These methods have so far only been applied to mosquito populations. However, they should have broad applicability to a range of taxa, although no assessment has yet been made on their performance under different dispersal conditions and study designs. Here we develop an R package and shiny app, kindisperse, that can be used to estimate dispersal parameters from the spatial distribution of close kin. kindisperse can handle study designs that target different life stages and allows for a range of dispersal kernel shapes and organismal life histories; we provide implementation examples for a vertebrate (Antechinus) and an invertebrate (Aedes). We use simulations run in kindisperse to compare the performance of two published close kin methodologies, showing that one method produces unbiased estimates whereas the other produces downward-biased estimates. We also use kindisperse simulations to investigate how study design affects dispersal estimates, and we provide guidelines for the size and shape of sample sites as well as the number of close kin needed for accurate estimates. kindisperse is easily adaptable for application to a variety of research contexts ranging from invasive pests to threatened species where noninvasive DNA sampling can be used to detect close kin.

A simple sexing test for elephant species and its application to faecal DNA.

We developed a novel real-time PCR assay for rapid sexing in all three elephant species, which amplifies small fragments of the orthologous sexual chromosome zinc finger protein genes ZFX/ZFY (65 bp). This assay is a simple, inexpensive and reliable tool that is suitable for non-invasive DNA samples and can be incorporated into larger SNP panels for individual identification and population genetic studies.

Eggshell membrane for DNA sexing of the endangered Maleo (Macrocephalon maleo)

Background: Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. However, its application in megapodes is still lacking. The previous genetic studies on megapodes used either blood or fresh tissue. Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo ( Macrocephalon maleo). Methods: We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Two different DNA extraction methods: alkaline lysis method and gSYNC TM DNA Extraction Kit were applied. To determine the sex of Maleo, we utilized PCR-based DNA sexing using CHD genes, with the primer set 2550F/2718R. Results: We successfully extracted all samples; the mean sample concentration was 267.5 ng/µl (range 47-510.5 ng/µl) and samples were of high purity (A260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males. Conclusions: Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds.

Eggshell membrane for DNA sexing of the endangered Maleo (Macrocephalon maleo)

Background: Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. However, its application in megapodes is still lacking. The previous genetic studies on megapodes used either blood or fresh tissue. Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo (Macrocephalon maleo). Methods: We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Two different DNA extraction methods: alkaline lysis method and gSYNCTM DNA Extraction Kit were applied. To determine the sex of Maleo, we utilized PCR-based DNA sexing using CHD genes, with the primer set 2550F/2718R. Results: We successfully extracted all samples; the mean sample concentration was 267.5 ng/µl (range 47–510.5 ng/µl) and samples were of high purity (A260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males. Conclusions: Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds.

Eggshell membrane for DNA sexing of the endangered Maleo (Macrocephalon maleo)

Background: Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. However, its application in megapodes is still lacking. The previous genetic studies on megapodes used either blood or fresh tissue. Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo (Macrocephalon maleo). Methods: We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Two different DNA extraction methods: alkaline lysis method and gSYNCTM DNA Extraction Kit were applied. To determine the sex of Maleo, we utilized PCR-based DNA sexing using CHD genes, with the primer set 2550F/2718R. Results: We successfully extracted all samples; the mean sample concentration was 267.5 ng/µl (range 47–510.5 ng/µl) and samples were of high purity (A260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males. Conclusions: Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds.

Eggshell membrane for DNA sexing of the endangered Maleo ( Macrocephalon maleo)

Background: Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. However, its application in megapodes is still lacking. The previous genetic studies on megapodes used either blood or fresh tissue. Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo ( Macrocephalon maleo). Methods: We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Two different DNA extraction methods: alkaline lysis method and gSYNC TM DNA Extraction Kit were applied. To determine the sex of Maleo, we utilized PCR-based DNA sexing using CHD genes, with the primer set 2550F/2718R. Results: We successfully extracted all samples; the mean sample concentration was 267.5 ng/µl (range 47–510.5 ng/µl) and samples were of high purity (A260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males. Conclusions: Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds.