Multiple-tubes Approach Research Articles

"Guess Who's Coming to Dinner": Molecular Tools to Reconstruct multilocus Genetic Profiles from Wild Canid Consumption Remains.

Simple SummaryWolves and European wildcats are two iconic predator species that can live in overlapping ecological contexts and also share their habitats with their domestic free-ranging relatives, increasing the risk of anthropogenic hybridisation and its possible deleterious consequences. By applying a multidisciplinary approach, we morphologically and molecularly analysed the cat remains found in a canid faecal sample collected in a forested area of central Italy. Individual multilocus genotypes of both predator and prey were identified turning out to be, respectively, a wolf showing traces of dog ancestry at autosomal microsatellite loci and a domestic cat.Non-invasive genetic sampling is a practical tool to monitor pivotal ecological parameters and population dynamic patterns of endangered species. It can be particularly suitable when applied to elusive carnivores such as the Apennine wolf (Canis lupus italicus) and the European wildcat (Felis silvestris silvestris), which can live in overlapping ecological contexts and sometimes share their habitats with their domestic free-ranging relatives, increasing the risk of anthropogenic hybridisation. In this case study, we exploited all the ecological and genetic information contained in a single biological canid faecal sample, collected in a forested area of central Italy, to detect any sign of trophic interactions between wolves and European wildcats or their domestic counterparts. Firstly, the faecal finding was morphologically examined, showing the presence of felid hair and claw fragment remains. Subsequently, total genomic DNA contained in the hair and claw samples was extracted and genotyped, through a multiple-tube approach, at canid and felid diagnostic panels of microsatellite loci. Finally, the obtained individual multilocus genotypes were analysed with reference wild and domestic canid and felid populations to assess their correct taxonomic status using Bayesian clustering procedures. Assignment analyses classified the genotype obtained from the endothelial cells present on the hair sample as a wolf with slight signals of dog ancestry, showing a qi = 0.954 (C.I. 0.780–1.000) to the wolf cluster, and the genotype obtained from the claw as a domestic cat, showing a qi = 0.996 (95% C.I. = 0.982–1.000) to the domestic cat cluster. Our results clearly show how a non-invasive multidisciplinary approach allows the cost-effective identification of both prey and predator genetic profiles and their taxonomic status, contributing to the improvement of our knowledge about feeding habits, predatory dynamics, and anthropogenic hybridisation risk in threatened species.

A new approach combining forensic thresholds and a multiple-tubes approach to unravel false microsatellite profiles from cross-contaminated sample material

Contamination and degradation are known challenges for reliable genotyping, since they can cause, among other problems, false microsatellite profiles. In this study we described a method to decrease the proportion of false microsatellite profiles from fish scale samples of endangered allis shads (Alosa alosa) from a reintroduction program, where cross-contamination with DNA from other individuals and potentially degradation of samples occurred. To maximize the portion of reliably measurable results, we modified and combined two known approaches—thresholds used in forensic DNA analyses and a multiple-tubes approach. This combined approach increased reliable microsatellite profiles compared with single approaches. The forensic thresholds and the multiple-tubes approach increased the measurable results from 55 to 67% and 75%, respectively, whereas the combined approach accomplished an increase to 90%. This illustrates the potential of the combined approach for other studies with comparable problems or sample material.

Genetic Diversity of Historical and Modern Populations of Russian Cattle Breeds Revealed by Microsatellite Analysis.

Analysis of ancient and historical DNA has great potential to trace the genetic diversity of local cattle populations during their centuries-long development. Forty-nine specimens representing five cattle breeds (Kholmogor, Yaroslavl, Great Russian, Novgorod, and Holland), dated from the end of the 19th century to the first half of the 20th century, were genotyped for nine polymorphic microsatellite loci. Using a multiple-tube approach, we determined the consensus genotypes of all samples/loci analysed. Amplification errors, including allelic drop-out (ADO) and false alleles (FA), occurred with an average frequency of 2.35% and 0.79%, respectively. A significant effect of allelic length on ADO rate (r2 = 0.620, p = 0.05) was shown. We did not observe significant differences in genetic diversity among historical samples and modern representatives of Kholmogor and Yaroslavl breeds. The unbiased expected heterozygosity values were 0.726–0.774 and 0.708–0.739; the allelic richness values were 2.716–2.893 and 2.661–2.758 for the historical and modern samples, respectively. Analyses of FST and Jost’s D genetic distances, and the results of STRUCTURE clustering, showed the maintenance of a part of historical components in the modern populations of Kholmogor and Yaroslavl cattle. Our study contributes to the conservation of biodiversity in the local Russian genetic resources of cattle.

A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Experimental evaluation of genetic predator identification from saliva traces on wildlife kills

Identification of predators from saliva traces on game species and/or livestock kills is gaining increasing importance in wildlife management, particularly in areas where direct wildlife–human conflicts regularly occur. When the noninvasive sampling of hairs and scats is difficult, as with rare and elusive predators, saliva samples constitute a potentially useful source of DNA. To test the feasibility of this approach in obtaining an accurate genotype of the predator, we applied an experimental approach. Captive wolves (Canis lupus) and lynxes (Lynx lynx) were allowed to feed on freshly killed roe deer (Capreolus capreolus) pieces for 1min. After removal, pieces were sampled for saliva traces after 1, 24, and 48h. Microsatellite analysis revealed that error rates and amplification failure increased sharply over time. While samples collected after 1 and 24 h yielded > 83% complete genotypes, values dropped to < 50% for samples collected after 48h, of which 7% were incorrect even when consensus genotypes from 9 polymerase chain reactions were obtained. Our results stress the importance of rapid sampling after carcass detection, as well as implementing a multiple-tubes approach when using microsatellite markers for genetic predator identification based on saliva traces.

非侵襲的試料を用いたDNA分析

Noninvasive genetics is one of the tools for clarifying society and ecology of wild primates. Here, I reviewed the methods of DNA analysis using noninvasive genetics. Although various types of noninvasive samples such as feces, urine, hair, and saliva have been used in wild mammals including primates, fecal samples are most common. The success rate of DNA analysis using the surface of feces is higher than that using the whole or inner part of feces. Numerous protocols for preserving fecal samples have been proposed, and one of the important points is to collect comparatively small amount of feces irrespective of the protocols. Temperature and rainfall before the collection of feces have negative impacts on DNA analyses, while diets have little impacts. The best ways to extract DNA might be to use QIAamp DNA Stool Mini Kit (QIAGEN) or modified CTAB methods. Although hair samples from nests of great apes have been used as noninvasive genetics, we should be aware that quantity of DNA which is extracted from a single dropped hair is low. When we conduct PCR amplification for reliable microsatellite genotype using noninvasive samples, we should take measures for PCR inhibitors present in extracts and genotyping errors such as allelic dropouts and false alleles. Multiple-tubes approach is necessary for most noninvasive samples and multiplex PCR method is a good way to reduce genotyping costs. Although noninvasive genetics can give us the estimates of relatedness which is useful for studying social behavior, pairwise relatedness estimates using less than 20 microsatellite loci has been shown to be unreliable. Development of an easy method to give robust data on pairwise relatedness might be important to understand the effect of relatedness on social behavior in the wild primate groups which have limited information about kinship due to short research period.

A reliable, single-step method for gender determination in black rhinoceros from low-copy template DNA

We describe an accurate, single-step 5 0 exonu- clease assay for gender determination of black rhinoceros Diceros bicornis, which targets a single nucleotide poly- morphism in exon 4 of the sex-linked zinc finger homo- logues in this species. This simple, high-throughput method facilitates the multiple-tubes approach that is necessary when using genetic material derived from non-invasive samples such as faecal material.

Skin swabbing of amphibian larvae yields sufficient DNA for efficient sequencing and reliable microsatellite genotyping

Skin swabbing, a minimally invasive DNA sampling method recently developed on adult amphibians, was tested on larvae of fire salamanders (Salamandra salamandra). The quality and quantity of the sampled DNA was evaluated by (i) measuring DNA concentration in DNA extracts, (ii) sequencing part of the mtDNA cytochrome b gene (692 bp) and (iii) genotyping eight polymorphic nuclear microsatellite loci. The multiple-tubes approach was used for calculating allelic dropout (ADO) and false allele (FA) rates to evaluate the reliability of the genotypes. DNA extracts from tissue samples of road-killed individuals were included in the study as positive controls. Our results showed that skin swabs of fire salamander larvae can provide DNA in sufficient quantity and quality, as sequencing was successful and no allelic dropouts or false alleles were detected. This method, tested for the first time on amphibian larvae, has proven to be an efficient and reliable alternative to the controversial tail fin clipping procedure.

Lessons from the use of non-invasive genetic sampling as a way to estimate Eurasian otter population size and sex ratio

Given the difficulties in establishing population parameters of elusive animals in the wild by traditional methods, such as trapping, much attention has been given in recent years to non-invasive genetic sampling. Our work compared estimates of population size and sex ratio derived from genetic sampling with the known number and sex of animals released during an otter reintroduction and reports on the pitfalls and opportunities that may be encountered in studies of this kind. This study makes use of 121 samples of otter spraints (faeces) collected over 7 months during a reintroduction in the Upper Thames (UK) where a total of 17 otters was released in two consecutive phases. Spraints were processed with a multiple tubes approach and seven microsatellites were used. Of all collected samples, 19 % were complete for at least five loci, the minimum required for discrimination between individuals. Six out of nine of the otters that were released in the first phase were detected, four males and two females, while none of the otters released in the second phase was detected probably due to a combination of sampling pitfalls and otter behaviour. In particular, the specific sex (mostly females) and dominance composition (lower) of the individuals in the second release group may explain our failure to detect individuals in this group. Taken together, our results add further evidence that genetic sampling approaches represent a potentially accurate and non-invasive route to census populations of otters but that the sampling design should take into account factors like the sex ratio and dominance composition of the population in order to maximise detection and minimise error.

Molecular sexing of Neotropical otter (Lontra longicaudis) noninvasive samples

Noninvasive molecular assays allow the investigation of elusive, rare and/or endangered organisms, and are useful for estimates of population parameters. Sex determination of noninvasive samples provides important information of sex ratio, social structure and dispersal patterns. Still, such assays remain unavailable for most carnivore species, precluding assessments of their population biology. Here we report a standardized protocol for molecular sexing of Neotropical otter noninvasive samples collected in the field. A duplex PCR assay was employed using a multiple-tube approach, resulting in reliable gender determination for 95% of the samples and presenting a low rate (<4%) of misassignment. This assay should foster the development of molecular ecological studies of this little-known species, and likely also contribute to similar endeavors on related otters.

Determining microsatellite genotyping reliability and mutation detection ability: an approach using small-pool PCR from sperm DNA

Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 × 10(-2) to 2.2 × 10(-3) mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies.

Field-based evaluation of scat DNA methods to estimate population abundance of the spotted-tailed quoll (Dasyurus maculatus), a rare Australian marsupial

Context. DNA extracted non-invasively from remotely collected scat samples has been used successfully to enumerate populations of a few endangered mammal species. However, scat DNA surveys relying on scent-marking behaviours need to identify if age- or sex-specific variations or seasonal changes in scat scent-marking patterns affect population estimates. Furthermore, owing to the low quantity and quality of scat DNA, a thorough assessment of the technique is needed when it is applied to different species to ensure that individual identification is reliable. Aims. In the current study, microsatellite genetic profiles derived from 208 remotely collected scats of the spotted-tailed quoll (Dasyurus maculatus), a rare Australian marsupial carnivore, were compared with DNA profiles from tissue of 22 live-trapped individuals from the same study area to critically assess the reliability of the non-invasive method to estimate population abundance. Methods. Scat samples were collected at scent-marking sites over 4 consecutive months (April–July 2005), 7 weeks of which overlapped with the trapping program to allow direct comparisons of population estimates. Key results. Combining a multiple-tubes approach with error checking analyses provided reliable genetic tags and resulted in the detection of the majority of the live-trapped population (18 of 22 individuals). Ten additional individuals not known from trapping were also observed from scat DNA. A longer-term sampling regime was required for scats than for trapping to allow direct detection of a large proportion of the population and to provide a comparable population estimate. Critically, the 4-month scat collection period highlighted the importance of performing scat surveys during the mating season when scat scent marking is more frequent, and to avoid sex and age biases in scat marking patterns. Implications. Non-invasive scat DNA sampling methods that rely on scent-marking behaviours need to consider the duration of the sampling period and temporal differences in behaviours by the sexes and age groups to ensure that meaningful population estimates are achieved.

Genetic diversity and individual identification of reintroduced otters (Lutra lutra) in north-eastern Spain by DNA genotyping of spraints

During the last century, otter populations in the Mediterranean area of the Iberian Peninsula were dramatically reduced and disappeared in many localities. A reintroduction programme was established in north-eastern Spain (Muga and Fluvia basins and the “Aiguamolls de l’Emporda” wetlands), by releasing 42 otters from four different Iberian populations, between 1995 and 2002. In order to evaluate the success of the reintroduction programme, we investigated microsatellite variation in the native populations of released otters as well as in the population present in the release area in 2004. We used non-invasively collected samples as the DNA source to avoid disturbing the animals in the wild. Laboratory procedures included the screening of samples and a multiple-tubes approach to detect and correct genotyping errors. Our results show that founders have been replaced by descendants in the release area and the population is in Hardy-Weinberg equilibrium, with a 1:1 sex ratio. Western Iberian otters—representing two-thirds of the founder group and released earlier—have greatly contributed to the genetic composition of the current population. The genetic patterns of otters found in a basin north of the studied area suggest a common origin with the released population. We also detected in a few otters the presence of alleles not identified in the original founder group, and we shall discuss the possible origin of these alleles.

A simulation test of the effectiveness of several methods for error-checking non-invasive genetic data

Non-invasive genetic sampling (NGS) is becoming a popular tool for population estimation. However, multiple NGS studies have demonstrated that polymerase chain reaction (PCR) genotyping errors can bias demographic estimates. These errors can be detected by comprehensive data filters such as the multiple-tubes approach, but this approach is expensive and time consuming as it requires three to eight PCR replicates per locus. Thus, researchers have attempted to correct PCR errors in NGS datasets using non-comprehensive error checking methods, but these approaches have not been evaluated for reliability. We simulated NGS studies with and without PCR error and ‘filtered’ datasets using non-comprehensive approaches derived from published studies and calculated mark–recapture estimates using CAPTURE. In the absence of data-filtering, simulated error resulted in serious inflations in CAPTURE estimates; some estimates exceeded N by[ges ]200%. When data filters were used, CAPTURE estimate reliability varied with per-locus error (Eμ). At Eμ=0.01, CAPTURE estimates from filtered data displayed <5% deviance from error-free estimates. When Eμ was 0.05 or 0.09, some CAPTURE estimates from filtered data displayed biases in excess of 10%. Biases were positive at high sampling intensities; negative biases were observed at low sampling intensities. We caution researchers against using non-comprehensive data filters in NGS studies, unless they can achieve baseline per-locus error rates below 0.05 and, ideally, near 0.01. However, we suggest that data filters can be combined with careful technique and thoughtful NGS study design to yield accurate demographic information.

DNA-Based Individual and Sex Identification from Wolverine (Gulo Gulo) Faeces and Urine

Non-invasive genetic analyses are important for studies of species that are rare, sensitive or at risk of extinction. This study investigates the possibility of using faeces and urine to obtain microsatellite genotypes for individual identification of wolverines (Gulo gulo). The reliability of the employed method was assessed by analysing independent amplifications of non-invasive samples (a multiple-tube approach) and by comparing genotypes obtained from faeces to genotypes obtained from blood or tissue of the same individual. Ten microsatellite markers were successfully amplified in 65% of the faecal samples (n = 32) and 40% of the urine samples (n = 22). Allelic dropout was found in 12 and 14% of the amplifications from extracts of faeces and urine, respectively. Nevertheless, all multi-locus genotypes were correct, as judged from comparison to data from tissue or blood samples, after three replicates. These results suggest that a non-invasive approach based on DNA-analysis of faeces can be a powerful tool in population monitoring of wolverines, potentially providing reliable estimates of population size and immigration rate. A second objective of the study was to develop markers for DNA-based sex identification in wolverines using non-invasive samples. We developed two Y-linked markers, one that was specific to wolverine and one that also successfully identified sex in another mustelid. Importantly, none of the markers amplified potential prey species such as reindeer or rodents.

Reliable microsatellite genotyping of dolphin DNA from faeces

AbstractNoninvasive samples have proved useful in genotyping studies of free‐ranging mammals. However, potential genotyping errors associated with such samples dictate the need for validation studies. This pilot study demonstrates the use of dolphin faeces in multilocus microsatellite genotyping studies. An empirical approach to calculating the rate of genotyping error was applied to data from matched pairs of blood or tissue and faecal samples from both captive and wild bottlenose dolphins. Microsatellite genotypes were assigned to dolphin faecal extracts with greater than 95% confidence by using a multiple tube approach, and at least two independent replicate genotypings.

Quantitative detection of t(14;18)-positive cells by real-time quantitative PCR using fluorogenic probes.

To detect t(14;18)-positive cells present in human lymphoma tissue, bone marrow aspirates and peripheral blood mononuclear cells (PBMNC), we have established an automated, real-time quantitative PCR using double-labeled fluorogenic probes. In relation to t(14;18)-positive genomic DNA or a cloned t(14;18)-DNA fragment, highly reproducible results can be obtained with initial copy numbers between 10 to 10(5). The detection of single copies has been verified by the stochastic multiple-tube approach. PBMNC cells obtained during clinical follow-up of patients with follicular lymphoma were analyzed by the one-step, real-time quantitative PCR and a two-step, semi-nested PCR combined with a limiting dilution assay. The quantitative results obtained by both assays correlate very well. Real-time quantitative PCR has several advantages: (i) it involves less critical pipetting steps, (ii) is less time-consuming and (iii) UTP, in combination with uracil-N-glycosylase, can be used to control carryover contamination. The higher specificity is due to optimized primer annealing conditions and MgCl2 concentration and the use of AmpliTaq Gold. The sensitivity is at least as high as by the two-step PCR. Real-time quantitative PCR will be very helpful in large epidemiological studies and in research for molecular staging and the detection of minimal residual tumor cells, including the analysis of blood stem-cell preparations to be used for transplantation after myelo-ablative therapy.

Plucked hair samples as a source of DNA: reliability of dinucleotide microsatellite genotyping.

To test whether plucked hairs are a reliable source of DNA for genotyping microsatellite loci, we carried out experiments using one, three, or 10 hairs per extract for 50 alpine marmots. For each extract, seven independent genotypings were performed for the same locus (multiple-tubes approach). Two types of genotyping errors were recorded: a false homozygote defined as the detection of only one allele of a true heterozygote, and a false allele defined as a PCR-generated allele that was not one of the alleles of the true genotype. Using DNA extracted from one, three, or 10 hairs the overall error rate was 14.00%, 4.86%, and 0.29%, respectively. Based on our results, we conclude that 10 hairs should be used to obtain consistently reliable genotypings using the single-tube approach, and that a single plucked hair could represent a reliable source of DNA if the multiple-tubes approach is used. For future studies of dinucleotide repeat diversity using DNA extracted from one to three shed or plucked hairs, we strongly recommend initiating an appropriate pilot study to quantify the error rate and to determine the reliability of the single-tube approach.

Noninvasive genetic tracking of the endangered Pyrenean brown bear population

Pyrenean brown bears Ursus arctos are threatened with extinction. Management efforts to preserve this population require a comprehensive knowledge of the number and sex of the remaining individuals and their respective home ranges. This goal has been achieved using a combination of noninvasive genetic sampling of hair and faeces collected in the field and corresponding track size data. Genotypic data were collected at 24 microsatellite loci using a rigorous multiple-tubes approach to avoid genotyping errors associated with low quantities of DNA. Based on field and genetic data, the Pyrenean population was shown to be composed at least of one yearling, three adult males, and one adult female. These data indicate that extinction of the Pyrenean brown bear population is imminent without population augmentation. To preserve the remaining Pyrenean gene pool and increase genetic diversity, we suggest that managers consider population augmentation using only females. This study demonstrates that comprehensive knowledge of endangered small populations of mammals can be obtained using noninvasive genetic sampling.

Reliable genotyping of samples with very low DNA quantities using PCR.

Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.