Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit

Noninvasive genetic sampling of faecal pellets can be a valuable method for monitoring rare and cryptic wildlife populations, like the pygmy rabbit (Brachylagus idahoensis). To investigate this method's efficiency for pygmy rabbit monitoring, we evaluated the effect of sample age on DNA degradation in faecal pellets under summer field conditions. We placed 275 samples from known individuals in natural field conditions for 1–60 days and assessed DNA quality by amplifying a 294‐base‐pair (bp) mitochondrial DNA (mtDNA) locus and five nuclear DNA (nDNA) microsatellite loci (111–221 bp). DNA degradation was influenced by sample age, DNA type, locus length and rabbit sex. Both mtDNA and nDNA exhibited high PCR success rates (94.4%) in samples <1 day old. Success rates for microsatellite loci declined rapidly from 80.0% to 42.7% between days 5 and 7, likely due to increased environmental temperature. Success rates for mtDNA amplification remained higher than nDNA over time, with moderate success (66.7%) at 21 days. Allelic dropout rates were relatively high (17.6% at <1 day) and increased to 100% at 60 days. False allele rates ranged from 0 to 30.0% and increased gradually over time. We recommend collecting samples as fresh as possible for individual identification during summer field conditions. Our study suggests that this method can be useful for future monitoring efforts, including occupancy surveys, individual identification, population estimation, parentage analysis and monitoring of genetic diversity both of a re‐introduced population in central Washington and across their range.     

Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA‐based mark–recapture analysis of Sonoran pronghorn

Knowledge of population demographics is important for species management but can be challenging in low‐density, wide‐ranging species. Population monitoring of the endangered Sonoran pronghorn (Antilocapra americana sonoriensis) is critical for assessing the success of recovery efforts, and noninvasive DNA sampling (NDS) could be more cost‐effective and less intrusive than traditional methods. We evaluated faecal pellet deposition rates and faecal DNA degradation rates to maximize sampling efficiency for DNA‐based mark–recapture analyses. Deposition data were collected at five watering holes using sampling intervals of 1–7 days and averaged one pellet pile per pronghorn per day. To evaluate nuclear DNA (nDNA) degradation, 20 faecal samples were exposed to local environmental conditions and sampled at eight time points from one to 124 days. Average amplification success rates for six nDNA microsatellite loci were 81% for samples on day one, 63% by day seven, 2% by day 14 and 0% by day 60. We evaluated the efficiency of different sampling intervals (1–10 days) by estimating the number of successful samples, success rate of individual identification and laboratory costs per successful sample. Cost per successful sample increased and success and efficiency declined as the sampling interval increased. Results indicate NDS of faecal pellets is a feasible method for individual identification, population estimation and demographic monitoring of Sonoran pronghorn. We recommend collecting samples >7 days old and estimate that a sampling interval of 4–7 days in summer conditions (i.e. extreme heat and exposure to UV light) will achieve desired sample sizes for mark–recapture analysis while also maximizing efficiency.     

Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize,Central America using cross‐species microsatellites and faecal DNA

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.     

基于粪便DNA的贺兰山岩羊亲权鉴定和婚配制研究

2017年7-8月和11-12月在贺兰山岩画风景区采集到369份岩羊新鲜粪便样本,用多态性较高的10个微卫星位点进行基因分型,共识别出282只岩羊个体。通过复合扩増和牙釉蛋白基因PCR法共鉴定出152只雄性,130只雌性。微卫星分析得到平均等位基因（Na）为10.1,平均观测杂合度（Ho）为0.7737,平均期望杂合度（He）为0.6512,平均多态信息（PIC）为0.6129。运用Cervus3.0软件进行亲权鉴定,并用Kingroup V2计算个体间的亲缘系数（r）,作为亲权鉴定的补充,共鉴定出父-母-子7对,父-子21对,母-子19对。根据亲缘关系对岩羊的婚配制进行推测分析,表明岩羊婚配制应属一雄多雌。     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

Detection of parasitizing coccidia and determination of host crane species, sex and genotype by faecal DNA analysis

In Japan, the three main crane species are the endangered red-crowned crane (Grus japonensis) inhabiting Hokkaido, the northernmost island of Japan; the vulnerable hooded crane (Grus monacha); and the vulnerable white-naped crane (Grus vipio). Both the hooded and white-naped cranes migrate in winter to Izumi in Kyushu, the southern island of Japan. In this study, we investigated the cranes and their coccidian parasites, through a targeted molecular approach using faecal DNA to develop a noninvasive method for infectious disease research. To determine the origin of noninvasively collected faecal samples, host species were identified by sequencing a region of approximately 470 bp of the mitochondrial 16S ribosomal RNA gene in the faecal DNA. Furthermore, to avoid sample redundancy, individual determination was performed by fragment analysis using microsatellite and sex-linked markers. For microsatellite genotyping, previously reported markers and markers isolated in this study were examined, and seven loci for red-crowned cranes, eight for hooded cranes and six for white-naped cranes displayed polymorphisms. A low error rate was demonstrated by comparing microsatellite data generated from faecal DNA samples with that generated from feather DNA samples, indicating a high reliability. Polymerase chain reaction-based capillary electrophoresis (PCR-CE), employing genetic markers in the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA, was employed to detect crane coccidia. The sensitivity of detection of PCR-CE using faecal DNA was inferior to that with traditional microscopy; however, our results suggest that PCR-CE can depict crane coccidia diversity with higher resolution and it is a useful tool to characterize community composition of coccidia in detail.     

Long‐term persistence of horse fecal DNA in the environment makes equids particularly good candidates for noninvasive sampling

Fecal DNA collected noninvasively can provide valuable information about genetic and ecological characteristics. This approach has rarely been used for equids, despite the need for conservation of endangered species and management of abundant feral populations. We examined factors affecting the efficacy of using equid fecal samples for conservation genetics. First, we evaluated two fecal collection methods (paper bag vs. ethanol). Then, we investigated how time since deposition and month of collection impacted microsatellite amplification success and genotyping errors. Between May and November 2014, we collected feral horse fecal samples of known age each month in a feral horse Herd Management Area in western Colorado and documented deterioration in the field with photographs. Samples collected and dried in paper bags had significantly higher amplification rates than those collected and stored in ethanol. There was little difference in the number of loci that amplified per sample between fresh fecal piles and those that had been exposed to the environment for up to 2 months (in samples collected in paper bags). After 2 months of exposure, amplification success declined. When comparing fresh (0–2 months) and old (3–6 months) fecal piles, samples from fresh piles had more matching genotypes across samples, better amplification success and less allelic dropout. Samples defecated during the summer and collected within 2 months of deposition had highest number of genotypes matching among samples, and lowest rates of amplification failure and allelic dropout. Due to the digestive system and amount of fecal material produced by equids, as well as their occurrence in arid ecosystems, we suggest that they are particularly good candidates for noninvasive sampling using fecal DNA.     

Molecular tracking of mountain lions in the Yosemite valley region in California: genetic analysis using microsatellites and faecal DNA

Twelve microsatellite loci were characterized in California mountain lions (Puma concolor) and sufficient polymorphism was found to uniquely genotype 62 animals sampled at necropsy. Microsatellite genotypes obtained using mountain lion faecal DNA matched those from muscle for all of 15 individuals examined. DNA from potential prey species and animals whose faeces could be misidentified as mountain lion faeces were reliably distinguished from mountain lions using this microsatellite panel. In a field application of this technique, 32 faecal samples were collected from hiking trails in the Yosemite Valley region where seven mountain lions previously had been captured, sampled, and released. Twelve samples yielded characteristic mountain lion genotypes, three displayed bobcat-type genotypes, and 17 did not amplify. The genotype of one of the 12 mountain lion faecal samples was identical to one of the mountain lions that previously had been captured. Three of the 12 faecal samples yielded identical genotypes, and eight new genotypes were detected in the remaining samples. This analysis provided a minimum estimate of 16 mountain lions (seven identified by capture and nine identified by faecal DNA) living in or travelling through Yosemite Valley from March 1997 to August 1998. Match probabilities (probabilities that identical DNA genotypes would be drawn at random a second time from the population) indicated that the samples with identical genotypes probably came from the same mountain lion. Our results demonstrate that faecal DNA analysis is an effective method for detecting and identifying individual mountain lions.     

Genotyping faeces of red pandas (<Emphasis Type="Italic">Ailurus fulgens</Emphasis>): implications for population estimation

The red panda (Ailurus fulgens) is an endangered species distributed in the Himalaya and Hengduan Mountains and extremely difficult to monitor because it is elusive, wary and nocturnal. However, recent advances in noninvasive genetics are allowing conservationists to indirectly estimate population size of this animal. Here, we present a pilot study of individual identification of wild red pandas using DNA extracted from faeces. A chain of optimal steps in noninvasive studies were used to maximize genotyping success and minimize error rate across sampling, selection of microsatellite loci, DNA extraction and amplification and data checking. As a result, 18 individual red pandas were identified successfully from 33 faecal samples collected in the field using nine red panda-specific microsatellite loci with a low probability of identity of 1.249 × 10⁻³ for full siblings. Multiple methods of tracking genotyping error showed that the faecal genetic profiles possessed very few genotyping errors, with an overall error rate of 1.12 × 10⁻⁵. Our findings demonstrate the feasibility and reliability of using faeces as an effective source of DNA for estimating and monitoring wild red panda populations.     

A novel method for collection and preservation of faeces for genetic studies

Quantitative and qualitative measurements of DNA were used to compare faecal sample storage in ethanol and silica with a novel method (two‐step) in which samples are soaked in ethanol and then desiccated with silica. Silica‐preserved samples had the lowest DNA concentrations. The two‐step method yielded significantly more DNA in high quality samples (average DNA concentrations > 100 pg/µL with all storage methods). However, for lower quality samples, the ethanol and two‐step methods performed similarly. The amounts and rates of sample degradation were not strongly affected by storage method and neither was the percentage of target DNA (< 1%) obtained from the samples.     

Empirical evaluation of preservation methods for faecal DNA

We evaluate the relative effectiveness of four methods for preserving faecal samples for DNA analysis. PCR assays of fresh faecal samples collected from free-ranging baboons showed that amplification success was dependent on preservation method, PCR-product size, and whether nuclear or mitochondrial DNA was assayed. Storage in a DMSO/EDTA/Tris/salt solution (DETs) was most effective for preserving nuclear DNA, but storage in 70% ethanol, freezing at –20°C and drying performed approximately equally well for mitochondrial DNA and short (<200 bp) nuclear DNA fragments. Because faecal DNA is diluted and degraded, repeated extractions from faeces may be necessary and short nuclear markers should be employed for genotyping. A review of molecular scatology studies further suggests that three to six faeces per individual should be collected.     

Leader of the pack: faecal pellet deposition order impacts PCR amplification in wombats

DNA sourced from faeces is notoriously less reliable than that from tissue. Hence, understanding whether faecal pellet quality varies within faecal piles may be important for sample selection. We hypothesized that the order in which faecal pellets are deposited may influence microsatellite polymerase chain reaction (PCR) amplification success from sampled faeces, more specifically, that first pellets deposited will have signatures of greater success than later ones. In a first test of the hypothesis, first and later-deposited pellets, as determined from the direction of footprints, were collected from fresh (overnight) faecal piles of northern hairy-nosed wombats (Lasiorhinus krefftii). DNA extracts were typed for seven microsatellite loci. We found that faecal deposition order significantly affected optical density of bands on autoradiographs (a measure of PCR amplification success) when the first faecal pellet was compared with the last one, but not when the first pellet was only distinguishable from later ones. The absence of a difference in amplification rate between first and later pellets is likely a reflection of the overall high amplification success in this study. That first pellets deposited yield more product suggests they contain more intestinal cells. Although further comparisons are needed, these results may inform sample selection in species for which success of microsatellite PCR amplification of faecal DNA is low. Deposition order may have more of an impact on amplification success and genotyping errors as faecal age increases.     

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.     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

Background

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

Methods

Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.

Results

No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.

Conclusions

Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.     

Improved Methods of Carnivore Faecal Sample Preservation,DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

Background

Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase.

Methods

We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval.

Results

Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively.

Conclusions

Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.     

Noninvasive methodology for the sampling and extraction of DNA from free‐ranging Atlantic spotted dolphins (Stenella frontalis)

Genetic sampling and molecular investigations are important parts of studying wild populations. However, collecting tissues from free‐ranging animals can be difficult or impractical. This study develops a sampling and extraction protocol for template DNA from faecal material collected in a marine environment from small cetaceans. DNA was extracted from faecal material of free‐ranging Atlantic spotted dolphins (Stenella frontalis) and subsequently tested for its suitability in molecular investigations by amplifying both mitochondrial and nuclear DNA. The resulting mitochondrial sequences were found to closely match known S. frontalis haplotypes. Three microsatellite loci were amplified and fall within the expected size range for cetaceans. Mother and calf families previously assigned by observation were genetically confirmed using both mitochondrial haplotype and allele sharing between the mother and offspring. The protocol effectively collects and extracts dolphin DNA from faecal samples and enables species identification as well as confirmation of genetic relatedness and should be considered as a noninvasive alternative to current protocols.     

Isolation and characterization of polymorphic microsatellite loci from Yellowcheek (Elopichthys bambusa)

Nine yellowcheek (Elopichthys bambusa) microsatellite loci were isolated using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Three to eight alleles per locus were detected in 29 samples collected from five populations of E. bambusa. The mean number of alleles was 5.6 ± 1.9 and the level of observed heterozygosities ranged from 0.415 to 0.843. These are the first microsatellite loci characterized from E. bambusa that can be used for estimating genetic diversity, population structure and parentage analysis.     

不同保存方法对川金丝猴粪便DNA提取效果的影响

目的:川金丝猴(Rhinopithecus roxellana)是我国特有珍稀物种,其粪便作为一种非损伤性样品,为珍稀濒危动物的种群数量调查、遗传多样性评价、亲缘关系、系统进化等研究带来了很大便利,本研究试图建立高效、简便的粪便样品保存方法。方法:在现有珍稀濒危动物粪便样品保存方法的基础上,分别使用干燥法、冷冻法和干燥-冷冻法保存川金丝猴的粪便样品,比较了不同保存方法的DNA提取效果,以及对mtDNA控制区片段的PCR扩增成功率和微卫星基因的PCR扩增效率。结果:干燥法、冷冻法和干燥-冷冻法三种不同保存方法保存粪便1周时间后,提取的粪便DNA样本扩增mtDNA片段的成功率均为92%,微卫星基因的扩增成功率分别为79%、78%、80%;保存2个月后,mtDNA片段扩增成功率分别为80%、76%和80%,微卫星基因扩增成功率分别为65%、61%、67%;保存6个月后,mtDNA片段扩增成功率分别为56%、52%和64%,微卫星基因扩增成功率分别40%、34%、46%。因此,随着保存时间的增长,三种方法的保存效率都将明显降低,但干燥-冷冻法得到的DNA样本扩增成功率相对较高。结论:粪便样品能够为川金丝猴的遗传多样性评价等相关研究提供有效信息,干燥-冷冻法保存能够更为有效的保证DNA的提取和基因扩增效率。     

DNA degradation in fish: Practical solutions and guidelines to improve DNA preservation for genomic research

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.