Non-invasive Source Of DNA Research Articles

De novo discovery of SNPs for genotyping endangered sun parakeets (Aratinga solstitialis) in Guyana

Parrots (Psittaciformes) are among the most endangered groups of birds today and remain threatened by habitat loss and exploitation for the live bird trade. Under such conditions, reliable and non-invasive monitoring techniques are crucial for successful conservation measures. In this study, we developed a panel of 86 high quality SNPs for genotyping endangered sun parakeets (Aratinga solstitialis) in Guyana, which form one of the last known breeding populations of this South American species in the wild. Genotyping was tested on different types of samples (blood, feathers, feces, beak and cloacal swabs). While blood performed best, feathers and feces also yielded reliable results and could thus be used as non-invasive sources of DNA for future population monitoring. Discriminant Analysis of Principal Components (DAPC) on genotypes revealed that Guyanese sun parakeets clustered separately from other psittacine species as well as conspecifics from a captive population. A priori known first-order kinships were also adequately detected by the SNP panel. Using a series of experimental contaminations, we found that contamination from other psittacine species and slight contamination (~ 10%) from conspecifics did not prevent successful genotyping and recognition of individuals. We show that instances of higher conspecific contamination (~ 50%) can be detected through an increased level of heterozygosity that falls outside the distribution of uncontaminated samples.

Saliva as a comparable-quality source of DNA for Whole Exome Sequencing on Ion platforms

BackgroundWhole Exome Sequencing (WES) utilises overlapping fragments prone to sequencing artefacts. Saliva, a non-invasive source of DNA, has been successfully used in WES studies on various platforms. This study explored the validity and quality of DNA sourced from saliva compared to whole blood on an Ion Platform. MethodsDNA was extracted from both sample types from four individuals. WES, performed on the Ion Proton platform was assessed for quality metrics (Depth, Genotyping Quality, etc.) and variant identification for the same source sample-pairs. ResultsNo significant differences in quality metrics were identified between data obtained from whole blood and saliva samples, with several saliva samples demonstrating higher coverage depth. Variants within the same sample, from the two genomic DNA sources, had an average concordance similar to other studies and platforms with different chemistry. ConclusionSaliva-extracted DNA provides comparable sequencing quality to whole blood for WES on Ion Torrent Platforms.

Improving our conservation genetic toolkit: ddRAD-seq for SNPs in snow leopards

Snow leopards (Panthera uncia) are an enigmatic, high-altitude species whose challenging habitat, low population densities and patchy distribution have presented challenges for scientists studying its biology, population structure, and genetics. Molecular scatology brings a new hope for conservation efforts by providing valuable insights about snow leopards, including their distribution, population densities, connectivity, habitat use, and population structure for assigning conservation units. However, traditional amplification of microsatellites from non-invasive sources of DNA are accompanied by significant genotyping errors due to low DNA yield and poor quality. These errors can lead to incorrect inferences in the number of individuals and estimates of genetic diversity. Next generation technologies have revolutionized the depth of information we can get from a species’ genome. Here we used double digest restriction-site associated DNA sequencing (ddRAD-seq), a well-established technique for studying non-model organisms, to develop a reference sequence library for snow leopards using blood samples from five Mongolian individuals. Our final data set reveals 4504 loci with a median size range of 221 bp. We identified 697 SNPs and low nucleotide diversity (0.00032) within these loci. However, the probability that two random individuals will share identical genotypes is about 10−168. We developed probes for DNA capture using this sequence library which can now be used for genotyping individuals from scat samples. Genetic data from ddRAD-seq will be invaluable for conducting population and landscape scale studies that can inform snow leopard conservation strategies.

Multifaceted DNA metabarcoding: Validation of a noninvasive, next-generation approach to studying bat populations.

As multiple species of bats are currently experiencing dramatic declines in populations due to white‐nose syndrome (WNS) and other factors, conservation managers have an urgent need for data on the ecology and overall status of populations of once‐common bat species. Standard approaches to obtain data on bat populations often involve capture and handling, requiring extensive expertise and unavoidably resulting in stress to the bats. New methods to rapidly obtain critical data are needed that minimize both the stress on bats and the spread of WNS. Guano provides a noninvasive source of DNA that includes information from the bat, but also dietary items, parasites, and pathogens. DNA metabarcoding is a high‐throughput, DNA‐based identification technique to assess the biodiversity of environmental or fecal samples. We investigated the use of multifaceted DNA metabarcoding (MDM), a technique combining next‐generation DNA sequencing (NGS), DNA barcodes, and bioinformatic analysis, to simultaneously collect data on multiple parameters of interest (bat species composition, individual genotype, sex ratios, diet, parasites, and presence of WNS) from fecal samples using a single NGS run. We tested the accuracy of each MDM assay using samples in which these parameters were previously determined using conventional approaches. We found that assays for bat species identification, insect diet, parasite diversity, and genotype were both sensitive and accurate, the assay to detect WNS was highly sensitive but requires careful sample processing steps to ensure the reliability of results, while assays for nectivorous diet and sex showed lower sensitivity. MDM was able to quantify multiple data classes from fecal samples simultaneously, and results were consistent whether we included assays for a single data class or multiple data classes. Overall, MDM is a useful approach that employs noninvasive sampling and a customizable suite of assays to gain important and largely accurate information on bat ecology and population dynamics.

Scale samples from Temminck’s ground pangolin (Smutsia temminckii): a non-invasive source of DNA

Non-invasive sampling aims to obtain suitable samples without causing harm to the study organism, thus making it a more preferred sampling technique. Molecular methods have advanced in such a way that quality DNA can be obtained from less invasive mammal samples. Various PCR enhancing techniques have also been developed to aid in amplification of low quality DNA samples, to produce reliable results that can be used to monitor populations and species or for forensic applications. Temminck’s ground pangolin is one of the few mammals to be covered in scales. In this study, we attempt to identify the most appropriate DNA isolation and PCR methods as well as determine whether PCR additives could be used to improve isolation of DNA and downstream amplification of mitochondrial DNA, from scale samples collected from these pangolins. In this study, it was determined that several different methods are required in order to successfully amplify DNA, and a decision tree rather than a single method should be followed.

Improved DNA profiles from aged horse feces using pressure cycling technology

Feces represent an easily available, non-invasive source of DNA. Often DNA extraction from feces is compounded with contamination, dietary inhibitors and primarily low quantity and poor quality of host DNA. In an effort to improve host DNA recovery from fecal samples, a new DNA extraction methods was developed. The objectives of this project was to use non-invasive sampling techniques of feces from domestic horses, provide a viable protocol for an improved DNA extraction, and genotype using six equine microsatellite on feces aged for 6 days. The technique used a modified Qiagen® DNA Stool Mini Kit protocol (Q) with pressure cycling technology (PCT). The DNA yield using PCT extraction was five fold greater as compared to the Q. The PCT mediated Qiagen (Q + PCT) technique yielded complete (six loci) equine DNA profiles of 100 % samples ≤2 days old, 90 % at 4 days and 60 % at 6 days post defecation. The results indicated that the Q + PCT increased DNA yield, and thereby increased the likelihood of obtaining an equine DNA profile from aged fecal samples. Using ML-RELATE analyses, the profiles obtained from the six equine loci could also be used for kinship analysis. Samples aged up to Day 6 as well as pasture samples with unknown days since defecation were successful in individualization of the contributors and could also be used to analyze kinship. The Q + PCT method should prove to be an extremely useful and reliable method for conservation or forensic cases where fecal matter may be the only sample available for analyses.

Habitat fragmentation and its implications for Endangered chimpanzeePan troglodytesconservation

AbstractTaraba State, Nigeria, is an important conservation site for the Endangered Nigeria–Cameroon chimpanzeePan troglodytes ellioti. Gashaka Gumti National Park, Nigeria's largest national park and home to potentially the largest contiguous population of the Nigeria–Cameroon chimpanzee, spans a significant portion of the eastern sector of Taraba and the adjoining Adamawa State. South of the Park, Ngel Nyaki Forest Reserve comprises two forest fragments and holds a small population of chimpanzees. We investigated the existence of patterns in population structure and dispersal within this region, using microsatellite loci extracted from non-invasive sources of DNA. Our results indicate that dispersal and thus gene flow between the groups of chimpanzees at the Park and Reserve is limited, at least more so than it is within the Park, and we identified a biased sex ratio at the Reserve, forewarning of potential conservation concerns in relation to demographic and genetic stochasticity. We discuss conservation actions that may be applicable to sustaining the population within Ngel Nyaki Forest Reserve.

Individual identification of endangered species using mosquito blood meals: a proof-of-concept study in Iberian lynx.

Host identification from mosquito blood meals has been routinely used to identify the feeding preferences of insects in studies on transmission of vector-borne pathogens. Here, we identified for the first time the susceptibility of the endangered Iberian lynx (Lynx pardinus) to the attack of a wild mosquito female, the mosquito Anopheles atroparvus. Furthermore, we used 11 microsatellite markers to test for the utility of vertebrate DNA isolated from insect blood meals for individual identification of wildlife. Only the three smallest markers were successfully amplified; however, this genotype did not match with any of the previously genotyped individuals in southern Spain. These results support the use of DNA from mosquito blood meals as a non-invasive source of DNA and a powerful tool on epidemiological and conservation biology studies. However, as may be the case of other non-invasive sampling methods, the utility of this technique is probably limited by the quantity and quality of vertebrate DNA.

A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Forensic dentistry in human identification: A review of the literature

An update is provided of the literature on the role of odontology in human identification, based on a PubMed-Medline search of the last 5 years and using the terms: “forensic dentistry” (n = 464 articles), “forensic odontology” (n = 141 articles) and “forensic dentistry identification” (n = 169 articles). Apart from these initial 774 articles, others considered to be important and which were generated by a manual search and cited as references in review articles were also included. Forensic dentistry requires interdisciplinary knowledge, since the data obtained from the oral cavity can contribute to identify an individual or provide information needed in a legal process. Furthermore, the data obtained from the oral cavity can narrow the search range of an individual and play a key role in the victim identification process following mass disasters or catastrophes. This literature search covering the last 5 years describes the novelties referred to buccodental studies in comparative identification, buccodental evaluation in reconstructive identification, human bites as a method for identifying the aggressor, and the role of DNA in dental identification. The oral cavity is a rich and noninvasive source of DNA, and can be used to solve problems of a social, economic or legal nature. Key words:Forensic identification, DNA, forensic dentistry, rugoscopy, cheiloscopy, saliva.

Coelomic fluid: a noninvasive source of DNA in earthworms

To investigate whether coelomic fluid secreted by earthworms can be a noninvasive source of DNA, we amplified and sequenced DNA extracted from the coelomic fluid and muscle tissue of eight worms. The sequences obtained using DNA extracted from both sources were identical. All cytochrome c oxidase subunit I (COI) mitochondrial DNA sequences, including those retrieved from GenBank, formed a monophyletic group of Metaphire sieboldi. The results indicate that we successfully extracted total DNA from coelomic fluid secreted by earthworm.

Incorporating Genotype Uncertainty into Mark–Recapture‐Type Models For Estimating Abundance Using DNA Samples

Sampling DNA noninvasively has advantages for identifying animals for uses such as mark-recapture modeling that require unique identification of animals in samples. Although it is possible to generate large amounts of data from noninvasive sources of DNA, a challenge is overcoming genotyping errors that can lead to incorrect identification of individuals. A major source of error is allelic dropout, which is failure of DNA amplification at one or more loci. This has the effect of heterozygous individuals being scored as homozygotes at those loci as only one allele is detected. If errors go undetected and the genotypes are naively used in mark-recapture models, significant overestimates of population size can occur. To avoid this it is common to reject low-quality samples but this may lead to the elimination of large amounts of data. It is preferable to retain these low-quality samples as they still contain usable information in the form of partial genotypes. Rather than trying to minimize error or discarding error-prone samples we model dropout in our analysis. We describe a method based on data augmentation that allows us to model data from samples that include uncertain genotypes. Application is illustrated using data from the European badger (Meles meles).

Precision of High-Throughput Single-Nucleotide Polymorphism Genotyping with Fingernail DNA: Comparison with Blood DNA

Recently, high-throughput single-nucleotide polymorphism (SNP) genotyping arrays have been used for genome-wide association study. Blood samples are commonly used for such analyses because they provide genomic DNA of high molecular weight and in high quantities. Blood sampling is often difficult, however, when patients are located a great distance from the laboratory and the sample requires careful handling for transportation. Instead, buccal swabs or urinary epithelial cells have been used as noninvasive sources of DNA(1). Fingernail clippings are also obtained noninvasively and more easily. Such samples need no special attention for transportation and can be preserved dry at room temperature for long periods; however, because fingernail clippings yield fragmented DNA that may be contaminated with certain PCR inhibitors(2), this sample type has seemed unsuitable for analyses requiring high-quality genomic DNA. In this study, we compared the precision of Affymetrix GeneChip™ array-based SNP genotyping with fingernail DNA vs blood DNA and demonstrated the usefulness of fingernail DNA in genotyping and genome copy-number analysis. The study protocol was approved by the Ethics Committee on Human Genome and Gene Analysis, Nagasaki University, and written informed consent was obtained from every volunteer. Ten milliliters of peripheral blood and 10 fingernail clippings were obtained from 5 healthy …

Genetic clues from olfactory cues: brown hyaena scent marks provide a non-invasive source of DNA for genetic profiling

Brown hyaena scent marks were tested for their potential to provide DNA suitable for PCR. Up to 100% of scent marks were successfully amplified. The approach can potentially be adapted to other species with similar pasting behaviour and scent mark morphology, or to samples that are environmentally exposed/degraded/inhibitor-rich or where there are very limited amounts of sample.

Practical applications for epigenetic biomarkers in cancer diagnostics

Cancer represents a major global health problem and improvement of cancer treatment requires the development of new and useful molecular diagnostic tests that enable the detection of occult tumors, direction of personalized treatments, monitoring of patients during therapeutic intervention and prediction of long-term clinical outcomes. The ideal molecular diagnostic for cancer testing will be based upon non-invasive sources of DNA and will employ biomarkers that have excellent sensitivity, specificity and overall predictive value. Numerous genes are known to be hypermethylated during cancer development and progression. These methylation-sensitive genes represent potentially valuable epigenetic biomarkers for development of practical cancer molecular diagnostics. In fact, many epigenetic biomarkers have proven to possess excellent predictive value in assays designed to detect occult (or developing) neoplasms and/or forecast clinical course/outcome. The progress to date in this emerging area of cancer diagnostics suggests that we are not far away from a time when testing for epigenetic biomarkers will represent an integral part of cancer screening protocols that can be effectively applied to the general population and/or to groups of people with defined risk factors for specific cancer types.

Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA

Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA

Pooling hair samples to increase DNA yield for PCR

Hairs are useful non-invasive sources of DNA,but the DNA yield can be very small, thus promoting genotyping errors. Using multiple hairs can counter this problem, but mayintroduce multiple contributors to a sample if collected remotely. With microsatellite genotyping, samples representing multiple animals are obvious if three or more alleles are detected at any locus: these samples canthen be removed from any analyses. However, some multiple-individual samples may have only one or two alleles at each of the loci examined. We investigated the probability of failing to identify mixed pooled samples by simulating pooled samples (10~000 replicates)from microsatellite data from the northern and southern hairy-nosed wombats (NHNW, Lasiorhinus krefftii; SHNW, L.latifrons), species with low and high genetic diversity respectively. The majority (81.7%)of the 40 000 simulated samples had three or four alleles, so were readily identified as mixed. In the remaining 1-or-2-allele SHNW samples, forensic science software (DNAMIX) correctly identified mixed versus single-individual samples for all cases when the probability of locus failure was low(P (LF) = 0.1), and 99% of samples whenlocus failure was high (P (LF) = 0.5). For NHNW however, the probability of failing to identify a mixed sample was too high for population size estimation (0.05), even when the probability of locus failure was low. Incases such as this, pooled samples may beadequate for less demanding tasks, such as estimation of allele proportions. However, for animal populations with at least average levels of genetic variation, pooling of samples could safely be utilised for most applications.

Determination of haemophilia A carrier status from hair samples using polymerase chain reaction technique

The usefulness of intragenic restriction fragment length polymorphisms (RFLPs) for BclI, HindIII and XbaI, adapted for polymerase chain reaction (PCR), was tested for the detection of haemophilia A carrier status in the consultant of a family in which only haematological information was available on the inheritance of the trait. Hair follicles were used as the non-invasive source of DNA. The mother was found to be homozygous for BclI and heterozygous for HindIII sites, whereas her status as regards informativeness could not be established for XbaI. On the basis of HindIII RFLP, the daughter was found to be a carrier of the haemophilia trait. This was confirmed by sequencing the amplified intron 19 of the mother and the daughter. The RFLP for XbaI did not appear to be suitable for carrier detection using PCR due to the difficulty of establishing homozygosity or heterozygosity from the results of digestion of the amplified product.