Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays

Thaden, Alina von; Cocchiararo, Berardino; Jarausch, Anne; Jüngling, Hannah; Karamanlidis, Alexandros A.; Tiesmeyer, Annika; Nowak, Carsten; Muñoz-Fuentes, Violeta

doi:10.1038/s41598-017-10647-w

Cited by 67 publications

(79 citation statements)

References 83 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Using a more economical extraction kit could reduce the per-sample cost further. Because we observed no disagreement among replicates for samples with 100% call rates, costs could be reduced further by reducing the number of replicate PCRs as recommended by Von Thaden et al (2017). We did not include labor cost as these vary across institutions or primer development costs as these depend on the genomic information available for the focal species and are not a recurring cost.…”

Section: Discussionmentioning

confidence: 99%

“…Recent SNP genotyping approaches for noninvasive samples include the Fluidigm Dynamic Array platform (Fluidigm Corp) and the MassARRAY platform from Sequenom. The Fluidigm platform in particular has produced high genotyping success and low error rates for both fecal and hair samples (Kraus et al, 2015; Von Thaden et al, 2017). However, these techniques require specialized equipment often rendering them prohibitively expensive for wildlife research (Andrews, De Barba, Russello, & Waits, 2018; Carroll et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Eriksson

Ruprecht

Levi

2019

Preprint

View full text Add to dashboard Cite

5Non-invasive genotyping methods have become key elements of wildlife research over the last 6 two decades, but their widespread adoption is limited by high costs, low success rates, and high 7 error rates. The information lost when genotyping success is low may lead to decreased precision 8 in animal population densities which could misguide conservation and management actions. Single 9 nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used 10 microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to 11 genotyping errors, and produce results that are easily shared among laboratories. Here, we outline 12 a detailed protocol for cost-effective and accurate noninvasive SNP genotyping using highly 13 multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for 14 individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis 15 latrans). Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, 16 representing a substantial increase compared to previous microsatellite-based studies at a cost of 17 under $5 per PCR replicate (excluding labor). The accuracy of the genotypes was further 18 corroborated in that genotypes from scats matching known, GPS-collared coyotes were always 19 located within the territory of the known individual. We also show that different levels of 20 multiplexing produced similar results, but that PCR product cleanup strategies can have 21 2 substantial effects on genotyping success. By making noninvasive genotyping more affordable, 22 accurate, and efficient, this research may allow for a substantial increase in the use of 23 noninvasive methods to monitor and conserve free-ranging wildlife populations. 24 Keywords 25 Fecal DNA, individual identification, next-generation sequencing, noninvasive samples, single 26 nucleotide polymorphisms, SNP genotyping. 27 28 29 48 rates when using fragmented DNA from noninvasive sources (Morin, Luikart, & Wayne, 2004).49 Unlike microsatellite genotyping, which use size-based allele determination, SNP alleles are 50 provided directly in the DNA sequence, which facilitates automated allele calling and results that 51 are easily shared among laboratories and research groups.52 4 Recent SNP genotyping approaches for noninvasive samples include the Fluidigm 53 Dynamic Array platform (Fluidigm Corp) and the MassARRAY platform from Sequenom. The 54 Fluidigm platform in particular has produced high genotyping success and low error rates for 55 both fecal and hair samples (Kraus et al., 2015; Von Thaden et al., 2017). However, these 56 techniques require specialized equipment often rendering them prohibitively expensive for 57 wildlife research (Andrews, De Barba, Russello, & Waits, 2018; Carroll et al., 2018). A largely 58 unexplored strategy is to amplify many SNPs together in multiplex PCR and sequence them 59 using high-throughput sequencers. Such genotyping by amplicon sequencing (GBAS) is both 60 cost-effective, a...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Eriksson

Ruprecht

Levi

2019

Preprint

View full text Add to dashboard Cite

show abstract

“…The low error rate is important for all aspects of molecular ecology, but particularly for inferences of individual identification, parentage and relatedness. In addition, the Fluidigm platform had a higher genotyping success rate than microsatellite genotyping in hair samples from wolf faeces (87% and 70%, respectively) and wild cat hair (80% vs. 54%) but similar success rates in brown bear hair samples (97% and 99%, respectively; after quality control; von Thaden et al., ).…”

Section: Sampling and Methodological Considerationsmentioning

confidence: 96%

Genetic and genomic monitoring with minimally invasive sampling methods

Carroll

Bruford

DeWoody

et al. 2018

Evolutionary Applications

146

108

View full text Add to dashboard Cite

The decreasing cost and increasing scope and power of emerging genomic technologies are reshaping the field of molecular ecology. However, many modern genomic approaches (e.g., RAD‐seq) require large amounts of high‐quality template DNA. This poses a problem for an active branch of conservation biology: genetic monitoring using minimally invasive sampling (MIS) methods. Without handling or even observing an animal, MIS methods (e.g., collection of hair, skin, faeces) can provide genetic information on individuals or populations. Such samples typically yield low‐quality and/or quantities of DNA, restricting the type of molecular methods that can be used. Despite this limitation, genetic monitoring using MIS is an effective tool for estimating population demographic parameters and monitoring genetic diversity in natural populations. Genetic monitoring is likely to become more important in the future as many natural populations are undergoing anthropogenically driven declines, which are unlikely to abate without intensive adaptive management efforts that often include MIS approaches. Here, we profile the expanding suite of genomic methods and platforms compatible with producing genotypes from MIS, considering factors such as development costs and error rates. We evaluate how powerful new approaches will enhance our ability to investigate questions typically answered using genetic monitoring, such as estimating abundance, genetic structure and relatedness. As the field is in a period of unusually rapid transition, we also highlight the importance of legacy data sets and recommend how to address the challenges of moving between traditional and next‐generation genetic monitoring platforms. Finally, we consider how genetic monitoring could move beyond genotypes in the future. For example, assessing microbiomes or epigenetic markers could provide a greater understanding of the relationship between individuals and their environment.

show abstract

“…We aimed to generate a lynx SNP-panel with 96 SNPs for highthroughput genotyping using Fluidigm's SNPtype ™ assays (Fluidigm Corp., San Francisco, CA, USA), which has proven effective for SNP genotyping when using DNA extracted from noninvasively collected samples (see Von Thaden et al, 2017). Alternative high-throughput…”

Section: Snp-panel Developmentmentioning

confidence: 99%

“…The development of SNP-panels for wildlife species without available genomic resources relies on a preceding phase of SNP discovery. As SNP-panels utilized in genetic monitoring of wildlife species require only a limited number of markers-usually in the order of 24-384 SNPs (e.g., Bayerl et al, 2018;Fitak, Naidu, Thompson, & Culver, 2016;Kleinman-Ruiz et al, 2017;Kraus et al, 2015;Norman, Street, & Spong, 2013;Nussberger, Wandeler, & Camenisch, 2014; von Thaden et al, 2017)-high-throughput sequencing approaches that retrieve data for only a subset of the genome can be used because they yield sufficient genome-wide sequence variants. The most popular and widespread techniques used in population genetics and evolutionary biology are restriction site-associated DNA sequencing (RAD-seq; Miller, Dunham, Amores, Cresko, & Johnson, 2007) and variants thereof (e.g., Elshire et al, 2011;Kraus et al, 2011), whole-transcriptome shotgun sequencing (RNA-seq; Wang, Gerstein, & Snyder, 2009) and targeted capture (Gnirke et al, 2009;Olson, 2007).…”

Section: Introductionmentioning

confidence: 99%

Targeted resequencing of codingDNAsequences forSNPdiscovery in nonmodel species

Förster

Bull

Lenz

et al. 2018

Molecular Ecology Resources

Self Cite

View full text Add to dashboard Cite

Targeted capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L. lynx across the species' European range. A large set of biallelic candidate SNPs was then evaluated using a high-throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species.

show abstract

Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays

Cited by 67 publications

References 83 publications

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Genetic and genomic monitoring with minimally invasive sampling methods

Targeted resequencing of codingDNAsequences forSNPdiscovery in nonmodel species

Contact Info

Product

Resources

About