Amplification Success Rate Research Articles

CDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit.

BackgroundThe synthesis of complementary DNA (cDNA) for use in the detection of BCR-ABL1 at the Major Molecular Response (MMR) level is a well-established method used by clinical laboratories world-wide. However, the quality of cDNA provides sensitivity challenges and consequently affects the detection of Minimal Residual Disease (MRD).ResultsHerein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences.ConclusionsAccurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-015-0014-x) contains supplementary material, which is available to authorized users.

Evaluating the interaction of faecal pellet deposition rates andDNAdegradation rates to optimize sampling design forDNA‐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 four to seven 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 [Corrected].

Development and characterization of nine novel microsatellite markers for the milkfish Chanos chanos

The milkfish, Chanos chanos, is an important aquaculture resource in Southeast Asia. Using NGS data, 72 microsatellite markers were developed. PCR product confirmation using agarose gel electrophoresis allowed the identification of 24 utilizable markers. Milkfish samples (n = 48) from a hatchery stock in Palawan, Philippines were analyzed for genetic variability at the aforementioned 24 loci. Consequently, nine of these microsatellite loci were noted to have high success rate in amplification, exhibited polymorphism with 19 maximum number of alleles and no null alleles. These are the first microsatellite markers to be developed and characterized for C. chanos that will efficiently enable genetic stock delineation and monitoring as well as marker-aided genetic improvement research.

DNA barcoding Chinese medicinal Bupleurum

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China.

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear-cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH-psbA and trnL-F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra- and inter-specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH-psbA (100%), trnL-F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH-psbA and trnL-F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.

Procedures to genotype problematic non-invasive otter (Lutra lutra) samples

Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive and rare species such as the Eurasian otter (Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower than in other carnivores. We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0 % and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to distinguish the individual unrelated otters (PID = 0.001), while 13 loci were needed to distinguish sibling otters (PIDsibs = 0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success.

Microsatellite cross-species amplification and utility in southern African elasmobranchs: A valuable resource for fisheries management and conservation.

BackgroundSimilarly to the rest of the world, southern Africa’s diverse chondrichthyan fauna is currently experiencing high fishing pressures from direct and non-direct fisheries to satisfy market demands for shark products such as fins and meat. In this study, the development of microsatellite markers through cross-species amplification of primer sets previously developed for closely related species is reported as an alternative approach to de novo marker development. This included the design of four microsatellite multiplex assays and their cross-species utility in genetic diversity analysis of southern African elasmobranchs. As this study forms part of a larger project on the development of genetic resources for commercially important and endemic southern African species, Mustelus mustelus was used as a candidate species for testing these multiplex assays in down-stream applications.ResultsThirty five microsatellite primer sets previously developed for five elasmobranch species were selected from literature for testing cross-species amplification in 16 elasmobranch species occurring in southern Africa. Cross-species amplification success rates ranged from 28.6%-71.4%. From the successfully amplified microsatellites, 22 loci were selected and evaluated for levels of polymorphism, and four multiplex assays comprising of the 22 microsatellites were successfully constructed, optimised and characterised in a panel of 87 Mustelus mustelus individuals. A total of 125 alleles were observed across all loci, with the number of alleles ranging from 3–12 alleles. Cross-species amplification of the four optimised multiplex assays was further tested on 11 commercially important and endemic southern African elasmobranch species. Percentage of polymorphism ranged from 31.8%-95.5% in these species with polymorphic information content decreasing exponentially with evolutionary distance from the source species.ConclusionsCross-species amplification of the 35 microsatellites proved to be a time- and cost-effective approach to marker development in elasmobranchs and enabled the construction of four novel multiplex assays for characterising genetic diversity in a number of southern African elasmobranch species. This study successfully demonstrated the usefulness of these markers in down-stream applications such as genetic diversity assessment and species identification which could potentially aid in a more integrative, multidisciplinary approach to management and conservation of commercially important cosmopolitan and endemic elasmobranch species occurring in southern Africa.

Evaluation of fecal DNA preservation and extraction methods in Przewalski’s horse

The study compares the effects of three preservation methods (ethanol, drying and freezing) and two extraction methods (commercial kit and manual) on the PCR success rate and genotyping errors with fecal DNA from Przewalski’s horse (Equus przewalskii). The interaction term between preservation and extraction was significant, and the highest amplification success rate (0.661) was obtained with samples stored in ethanol and extracted manually. The preservation method and time significantly impacted microsatellite amplification success rates. The method of preservation had an effect on the rate of allelic dropout (ADO), but the rate of false alleles (FA) varied nonsignificantly. Feces preserved in ethanol yielded the lowest ADO and FA, followed by drying and freezing. Our optimized preservation protocol probably have wide applicability to equids and rhinocerotids that produce large, high-fiber fecal pellets.

Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens.

Forensic mitochondrial DNA (mtDNA) testing requires appropriate, high quality reference population data for estimating the rarity of questioned haplotypes and, in turn, the strength of the mtDNA evidence. Available reference databases (SWGDAM, EMPOP) currently include information from the mtDNA control region; however, novel methods that quickly and easily recover mtDNA coding region data are becoming increasingly available. Though these assays promise to both facilitate the acquisition of mitochondrial genome (mtGenome) data and maximize the general utility of mtDNA testing in forensics, the appropriate reference data and database tools required for their routine application in forensic casework are lacking. To address this deficiency, we have undertaken an effort to: (1) increase the large-scale availability of high-quality entire mtGenome reference population data, and (2) improve the information technology infrastructure required to access/search mtGenome data and employ them in forensic casework. Here, we describe the application of a data generation and analysis workflow to the development of more than 400 complete, forensic-quality mtGenomes from low DNA quantity blood serum specimens as part of a U.S. National Institute of Justice funded reference population databasing initiative. We discuss the minor modifications made to a published mtGenome Sanger sequencing protocol to maintain a high rate of throughput while minimizing manual reprocessing with these low template samples. The successful use of this semi-automated strategy on forensic-like samples provides practical insight into the feasibility of producing complete mtGenome data in a routine casework environment, and demonstrates that large (>2kb) mtDNA fragments can regularly be recovered from high quality but very low DNA quantity specimens. Further, the detailed empirical data we provide on the amplification success rates across a range of DNA input quantities will be useful moving forward as PCR-based strategies for mtDNA enrichment are considered for targeted next-generation sequencing workflows.

DNA barcoding assessment of green macroalgae in coastal zone around Qingdao, China

An assessment with assistance of DNA barcoding was conducted on green macroalgae in coastal zone around Qingdao, China, during the period of April–December, 2011. Three markers were applied in molecular discrimination, including the plastid elongation factor tufA gene, the internal transcribed spacer (ITS) region of the ribosomal cistron and rubisco large subunit gene 3′ regions (rbcL-3P). DNA barcoding discriminated 8 species, excluding species of genus Cladophora and Bryopsis due to failures in amplification. We ascertained and corrected 4 species identified by morphological methods for effectively assisting the classification. The gene tufA presented more advantages as an appropriate DNA marker with the strongest amplification success rate and species discrimination power than the other two genes. The poorest sequencing success largely handicapped the application of ITS. Samples identified by tufA and rbcL as Ulva flexuosa were clustered into the clade of U. prolifera by ITS in the neighbor-joining tree. Confusion with discrimination of the complex of U. linza, U. procera and U. prolifera (as the LPP complex) still existed for the three DNA markers. Based on our results, rbcL is recommended as a preferred marker for assisting tufA to discriminate green macroalgae. In distinguishing green-tide-forming Ulva species, the free-floating sample collected from the green tide in 2011 was proved to be identical with U. prolifera in Yellow Sea for ITS and rbcL genes. This study presents a preliminary survey of green macroalgae distributed in the coastal area around Qingdao, and proves that DNA barcoding is a powerful tool for taxonomy of green macroalgae.

Forensic animal DNA analysis using economical two-step direct PCR

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 ± 180.84 ng/μL. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire(®) Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community.

Short-read DNA Sequencing Yields Microsatellite Markers for Rheum

Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.

Shake and stew: a non‐destructive PCR‐ready DNA isolation method from a single preserved fish larva

A rapid non-destructive alternative to isolate DNA from an individual fish larva is presented, based on the suspension of epithelial cells through vortex forces, and the release of DNA in a heated alkaline solution. DNA from >6056 fish larvae isolated using this protocol has yielded a high PCR amplification success rate (>93%), suggesting its applicability to other taxonomic groups or sources when tissue amount is the limiting factor.

Application of DNA barcoding in Roscoea (Zingiberaceae) and a primary discussion on taxonomic status of Roscoea cautleoides var. pubescens

DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, three chloroplast markers (rbcL, trnH-psbA and matK) and one nrDNA marker (ITS) were tested for species identification in Roscoea. The ITS and trnH-psbA regions showed high success rate of PCR amplification and bidirectional sequencing, as well as perfect discriminatory ability. On the contrary, rbcL possessed no genetic variation and matK was relatively difficult in PCR amplification and DNA sequencing. Combination of multiple markers greatly improved identification ability of DNA barcoding. ITS + trnH-psbA could effectively discriminate 90% species based on method of the Neighbor-Joining (NJ) tree. Awful PCR amplification and DNA sequencing of matK restricted its efficiency, although it showed rich genetic variability in Roscoea. Moreover, it might be more appropriate to treat Roscoea cautleoides var. pubescens as an independent species based on molecular data, namely R. pubescens Z. Y. Zhu.

How to overcome genotyping errors in non-invasive genetic mark-recapture population size estimation-A review of available methods illustrated by a case study

The main goal of non-invasive genetic capture-mark-recapture (CMR) analysis is to gain an unbiased and reliable population size estimate of species that cannot be sampled directly. The method has becomean importantandwidelyusedtoolto research andmanage wildlifepopulations.However,researchers have to struggle with low amplification success rates and genotyping errors, which substantially bias subsequent analysis. To receive reliable results and to minimize the time and costs required for non-invasive microsatellite genotyping, one must carefully choose a species-specific sampling design, methods that maximize the amount of template DNA, and methods that could overcome genotyping errors, especially when using low-quality samples.This articlereviewsthe literature and theprosandcons ofthemain methods used along the process described above. The review is strengthened by a case study on Eurasian otters (Lutra lutra) using feces; we tested several methods for their appropriateness to accommodate for genotyping errors. Based on this method testing, we demonstrated that high genotyping error rates are the key problem in this process leading to a severely flawed dataset if no consensus genotype is formed. However, even if generating consensus genotypes minimizes errors dramatically, we show that it may not achieve a definite eradication of all errors, which results in overestimated population sizes if conventional estimators are used. In conjunction with these findings, we offer a step-by-step protocol for non-invasive genetic CMR studies to achieve a reliable estimate of population sizes in the presence of high genotyping error rates. 2013 The Wildlife Society.

Informative microsatellites for freshwater and marine shrimp species

Marine and freshwater shrimp are overexploited worldwide due to their high commercial value being usually fished in a predatory manner. The concern about conserving the stocks for economical or ecological reasons has been threatened by the absence of molecular markers for the majority of these species. In this work, we described ten anonymous microsatellites isolated from an enriched-library with magnetic beads technology of Penaeus (Litopenaeus) vannamei genome, considered the most economically important marine shrimp in the world. In addition, we tested these loci and a panel of 21 Simple Sequence Repeat from Expressed Sequence Tag (EST-SSR) loci, also isolated from P. vannamei, in ten other marine and two freshwater shrimp species. The cross species amplification success rate ranged from 32 to 77% in Macrobrachium jelskii and Penaeus (Litopenaeus) schmitti, respectively. These microsatellites appear conservative in a wide spectrum of shrimp groups, facilitating genetic studies in those species in which no microsatellites have been yet described. Key words: Conservative, Simple Sequence Repeat (SSR), shrimp genetic, penaeid, Macrobrachium.

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55% (11/20) for exon 19 deletion, 45% (9/20) for T790M, and 85% (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80% of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Mitochondrial and pedigree analysis in Przewalski's horse populations: implications for genetic management and reintroductions

Background and Aims: Przewalski’s horses have been imported from the western zoos to China since 1985. Yet the genetic diversity in China’s populations has not been studied, thus lacking of such knowledge inevitably affects this population’s management. The aim of this study was to assess genetic diversity in Chinese population of Przewalski’s horses via mitochondrial DNA (mtDNA) control region and pedigree analysis. Materials and methods: Two captive and one reintroduced populations were examined based on mitochondrial DNA control region variation via fecal sampling from 2010 to 2012, together with pedigree analysis. Results: Amplification success rates of fecal mtDNA were as high as 96.2% (93.8%–100%), and were higher for sample in winter than in summer and autumn. Two haplotypes were identified and shared among three populations, but the proportion of individuals with each haplotype varied among the three populations (FST = 0.10874, p = 0.00978). Haplotype diversity in the released population (0.153) was much lower than that in the two captive populations (0.4011 and 0.4966), in accordance with the direction of increase in probability of identity at the dam lines. Conclusion: Future concerns in Przewalski’s horse population management should emphasize on strict reproduction control to minimize inbreeding in captivity, followed by long-term genetic diversity guidelines and non-invasive monitoring in the reintroduction programmes.

High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.

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.