Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.     

Isolation and multiplex genotyping of polymorphic microsatellite DNA markers in the snakehead murrel, Channa striata

Seven polymorphic microsatellite loci were isolated and characterized for the snakehead murrel, Channa striata (Channidae), a valuable tropical freshwater fish species. Among 25 specimens collected from Kedah state in Malaysia, the number of alleles per locus ranged from 2 to 7. Observed and expected heterozygosities ranged from 0.120 to 0.880 and 0.117 to 0.698, respectively. A single locus (CS1-C07) was significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction. These novel markers would be useful for population genetic studies of the C. striata.     

Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair

In the context of a study of wild chimpanzees, Pan troglodytes verus, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n= 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/10 μL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.     

Using DNA from non-invasive samples to identify individuals and census populations: an evidential approach tolerant of genotyping errors

DNA extracted from hair or faeces shows increasing promise for censusing populations whose individuals are difficult to locate. To date, the main problem with this approach has been that genotyping errors are common. If these errors are not identified, counting genotypes is likely to overestimate the number of individuals in a population. Here, we describe an algorithm that uses maximum likelihood estimates of genotyping error rates to calculate the evidence that samples came from the same individual. We test this algorithm with a hypothetical model of genotyping error and show that this algorithm works well with substantial rates of genotyping error and reasonable amounts of data. Additional work is necessary to develop statistical models of error in empirical data.     

Isolation,characterization and multiplex genotyping of raccoon tetranucleotide microsatellite loci

We have isolated and characterized 12 polymorphic tetranucleotide microsatellite loci in the raccoon (Procyon lotor). Three multiplex panels comprising the 12 loci were developed and 80 individuals from southeastern and western Ontario were genotyped; allele sizes were assigned without difficulty. One locus isolated was identified as an X‐linked marker. The number of alleles per locus ranged from six to 25 with the average heterozygosity ranging from 0.674 to 0.925. These loci will be used to characterize raccoon population structure across North America, and the data used to further understand the spread of raccoon rabies.     

Description of microsatellite markers and genotyping performances using feathers and buccal swabs for the Ivory gull (Pagophila eburnea)

We report 22 new polymorphic microsatellites for the Ivory gull (Pagophila eburnea), and we describe how they can be efficiently co-amplified using multiplexed polymerase chain reactions. In addition, we report DNA concentration, amplification success, rates of genotyping errors and the number of genotyping repetitions required to obtain reliable data with three types of noninvasive or nondestructive samples: shed feathers collected in colonies, feathers plucked from living individuals and buccal swabs. In two populations from Greenland (n=21) and Russia (Severnaya Zemlya Archipelago, n=21), the number of alleles per locus varied between 2 and 17, and expected heterozygosity per population ranged from 0.18 to 0.92. Twenty of the markers conformed to Hardy-Weinberg and linkage equilibrium expectations. Most markers were easily amplified and highly reliable when analysed from buccal swabs and plucked feathers, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. Although DNA amplification success using single shed feathers was generally high, the genotypes obtained from this type of samples were prone to error and thus need to be amplified several times. The set of microsatellite markers described here together with multiplex amplification conditions and genotyping error rates will be useful for population genetic studies of the Ivory gull.     

gemini: software for testing the effects of genotyping errors and multitubes approach for individual identification

Improvements in the determination of individual genotypes from samples with low DNA quantity and quality are of prime importance in molecular ecology and conservation for reliable genetic individual identification (molecular tagging using microsatllites loci). Thus, errors (e.g. allelic dropout and false allele) appearing during samples genotyping must be monitored and eliminated as far as possible. The multitubes approach is a very effective but a costly and time‐consuming solution. In this paper, we present a simulation software that allows evaluation of the effect of genotyping errors on genetic identification of individuals and the effectiveness of a multitubes approach to correct these errors.     

A multi-samples, multi-extracts approach for microsatellite analysis of faecal samples in an arboreal ape

We investigated the effect of the number of faecal samples, ofextracts per sample and of PCRs per extract on the reliability ofgenotypes for a microsatellite locus in free-living orang-utans.For each individual 36 PCRs were performed using DNA extractionsfrom up to four faecal samples. We found a very largeinter-individual variation in positive PCRs (P+) (36/36 for oneindividual and 0/36 for another). As many as 30% of the cases ledto erroneous genotypes when only one P+ was obtained. It ispreferable to use at least 4 P+ per extract to reduce thisproportion to less than 1%. With 3 P+ results, erroneousgenotypes were still observed in 26% of the cases together. Theseresults indicate that it is necessary to do a minimum of 4 PCRsper extract. In order to have a chance to observe 4 P+, threeextracts should be ideally analysed for each sample. We alsorecommend that when possible two or more samples should becollected in the field to increase the chance of having extractscontaining DNA and to provide independent replicates. While werecognise the difficulty of working with faecal samples, weadvocate the use of faecal material for genetic studies ofcertain wild animal populations where the advantages of avoidingdisturbance, stress and injury are deemed of critical importance.     

Noninvasive population genetics: a review of sample source,diet, fragment length and microsatellite motif effects on amplification success and genotyping error rates

Noninvasive population genetics has found many applications in ecology and conservation biology. However, the technical difficulties inherent to the analysis of low quantities of DNA generally tend to limit the efficiency of this approach. The nature of samples and loci used in noninvasive population genetics are important factors that may help increasing the potential success of case studies. Here we reviewed the effects of the source of DNA (hair vs. faeces), the diet of focal species, the length of mitochondrial DNA fragments, and the length and repeat motif of nuclear microsatellite loci on genotyping success (amplification success and rate of allelic dropout). Locus-specific effects appeared to have the greatest impact, amplification success decreasing with both mitochondrial and microsatellite fragments’ length, while error rates increase with amplicons’ length. Dinucleotides showed best amplification success and lower error rates compared to longer repeat units. Genotyping success did not differ between hair- versus faeces-extracted DNA, and success in faeces-based analyses was not consistently influenced by the diet of focal species. While the great remaining variability among studies implies that other unidentified parameters are acting, results show that the careful choice of genetic markers may allow optimizing the success of noninvasive approaches.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

Locus effects and sources of error in noninvasive genotyping

In spite of more than a decade of research on noninvasive genetic sampling, the low quality and quantity of DNA in noninvasive studies continue to plague researchers. Effects of locus size on error have been documented but are still poorly understood. Further, sources of error other than allelic dropout have been described but are often not well quantified. Here we analyse the effects of locus size on allelic dropout, amplification success and error rates in noninvasive genotyping studies of three species, and quantify error other than allelic dropout.     

Reliable microsatellite genotyping of the Eurasian badger (Meles meles) using faecal DNA

The potential link between badgers and bovine tuberculosis has made it vital to develop accurate techniques to census badgers. Here we investigate the potential of using genetic profiles obtained from faecal DNA as a basis for population size estimation. After trialling several methods we obtained a high amplification success rate (89%) by storing faeces in 70% ethanol and using the guanidine thiocyanate/silica method for extraction. Using 70% ethanol as a storage agent had the advantage of it being an antiseptic. In order to obtain reliable genotypes with fewer amplification reactions than the standard multiple-tubes approach, we devised a comparative approach in which genetic profiles were compared and replication directed at similar, but not identical, genotypes. This modified method achieved a reduction in polymerase chain reactions comparable with the maximum-likelihood model when just using reliability criteria, and was slightly better when using reliability criteria with the additional proviso that alleles must be observed twice to be considered reliable. Our comparative approach would be best suited for studies that include multiple faeces from each individual. We utilized our approach in a well-studied population of badgers from which individuals had been sampled and reliable genotypes obtained. In a study of 53 faeces sampled from three social groups over 10 days, we found that direct enumeration could not be used to estimate population size, but that the application of mark-recapture models has the potential to provide more accurate results.     

TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.     

Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts

In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality samples was 96%, 24%, and 24% for microsatellite loci, and 98%, 97%, and 79% for SNPs, respectively. As measured by qPCR, good quality samples had a consistently high number of starting copies across all fragment sizes with little change between the smallest and largest size. In contrast, the intermediate and poor quality samples displayed decreases in starting copy number as fragment size increased, and was most pronounced with poor samples. Logistic regression of genotyping success by starting copy number indicated that in order to achieve at least 90% genotyping success, approximately 1,000 starting copies of nuclear DNA are necessary for microsatellite loci, and as few as 14 starting copies for SNP assays (but we recommend at least 50 copies to reduce genotyping error). While these guidelines apply specifically to Chinook salmon and the genetic markers included in this study, the principles are transferable to other species and markers due to the underlying process associated with template quantity and PCR amplification.     

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.