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219 results for “genotyping‐by‐sequencing”
Data from: Genotyping-by-sequencing for estimating relatedness in non-model organisms: avoiding the trap of precise bias
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Data from: Genotyping-in-Thousands by sequencing (GT-seq) panel development and application to minimally-invasive DNA samples to support studies in molecular ecology
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Data from: More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing
<p>Non-invasive genotyping methods have become key elements of wildlife research over the last two decades, but their widespread adoption is limited by high costs, low success rates, and high error rates. <span>The information lost when genotyping success is low may lead to decreased precision in animal population densities, which could misguide conservation and management actions.</span> <span>Single nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to genotyping errors, and produce results that are easily shared among laboratories.</span> Here, we outline a detailed protocol for cost-effective and accurate noninvasive SNP genotyping using multiplexed amplicon sequencing optimized for degraded DNA. <span>We validated this method for individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (<i>Canis latrans</i>) using 26 SNPs. </span><a name="_Hlk33181599">Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, representing a substantial increase compared to previous microsatellite-based studies while remaining at a low cost of under $5 per PCR replicate (excluding labor). </a>The accuracy of the genotypes was further corroborated in that genotypes from scats matching known, GPS-collared coyotes were always located within the territory of the known individual. We also show that different levels of multiplexing produced similar results, but that PCR product cleanup strategies can have substantial effects on genotyping success. By making noninvasive genotyping more affordable, accurate, and efficient, this research may allow for a substantial increase in the use of noninvasive methods to monitor and conserve free-ranging wildlife populations.</p>
Data from: Spontaneous hybridization and introgression between walleye (Sander vitreus) and sauger (S. canadensis) in two large reservoirs: insights from genotyping-by-sequencing
<p>Anthropogenic activities may facilitate undesirable hybridization and genomic introgression between fish species. Walleye (<i>Sander vitreus</i>) and sauger (<i>Sander canadensis</i>) are economically valuable freshwater species that can spontaneously hybridize in areas of sympatry. Levels of genomic introgression between walleye and sauger may be increased by modifications to waterbodies (e.g., reservoir development) and inadvertent propagation of hybrids in stocking programs. We used genotyping by sequencing (GBS) to examine 217 fish from two large reservoirs with mixed populations of walleye and sauger in Saskatchewan, Canada (Lake Diefenbaker, Tobin Lake). Analyses with 20,038 (r90) and 478 (r100) SNPs clearly resolved walleye and sauger, and classified hybrids with high confidence. F<sub>1</sub>, F<sub>2</sub>, and multi-generation hybrids were detected in Lake Diefenbaker, indicating potentially high levels of genomic introgression. In contrast, only F<sub>1</sub> hybrids were detected in Tobin Lake. Field classification of fish was unreliable; 7% of fish were misidentified based on broad species categories. Important for activities such as brood stock selection, 12/173 (7%) fish field-identified as pure walleye, and 1/24 (4%) identified as pure sauger were actually hybrids. In addition, 2/15 (13%) field-identified hybrids were actually pure walleye or sauger. We conclude that hybridization and introgression are occurring in Saskatchewan reservoirs, and that caution is warranted when using these populations in stocking programs. GBS offers a powerful and flexible tool for examining hybridization without pre-identification of informative loci, eliminating some of the key challenges associated with other marker types.</p>
Data from: Genotyping-by-sequencing provides the discriminating power to investigate the subspecies of Daucus carota (Apiaceae)
Background: The majority of the subspecies of Daucus carota have not yet been discriminated clearly by various molecular or morphological methods and hence their phylogeny and classification remains unresolved. Recent studies using 94 nuclear orthologs and morphological characters, and studies employing other molecular approaches were unable to distinguish clearly many of the subspecies. Fertile intercrosses among traditionally recognized subspecies are well documented. We here explore the utility of single nucleotide polymorphisms (SNPs) generated by genotyping-by-sequencing (GBS) to serve as an effective molecular method to discriminate the subspecies of the D. carota complex. Results: We used GBS to obtain SNPs covering all nine Daucus carota chromosomes from 162 accessions of Daucus and two related genera. To study Daucus phylogeny, we scored a total of 10,814 or 38,920 SNPs with a maximum of 10 or 30 % missing data, respectively. To investigate the subspecies of D. carota, we employed two data sets including 150 accessions: (i) rate of missing data 10 % with a total of 18,565 SNPs, and (ii) rate of missing data 30 %, totaling 43,713 SNPs. Consistent with prior results, the topology of both data sets separated species with 2n = 18 chromosome from all other species. Our results place all cultivated carrots (D. carota subsp. sativus) in a single clade. The wild members of D. carota from central Asia were on a clade with eastern members of subsp. sativus. The other subspecies of D. carota were in four clades associated with geographic groups: (1) the Balkan Peninsula and the Middle East, (2) North America and Europe, (3) North Africa exclusive of Morocco, and (4) the Iberian Peninsula and Morocco. Daucus carota subsp. maximus was discriminated, but neither it, nor subsp. gummifer (defined in a broad sense) are monophyletic. Conclusions: Our study suggests that (1) the morphotypes identified as D. carota subspecies gummifer (as currently broadly circumscribed), all confined to areas near the Atlantic Ocean and the western Mediterranean Sea, have separate origins from sympatric members of other subspecies of D. carota, (2) D. carota subsp. maximus, on two clades with some accessions of subsp. carota, can be distinguished from each other but only with poor morphological support, (3) D. carota subsp. capillifolius, well distinguished morphologically, is an apospecies relative to North African populations of D. carota subsp. carota, (4) the eastern cultivated carrots have origins closer to wild carrots from central Asia than to western cultivated carrots, and (5) large SNP data sets are suitable for species-level phylogenetic studies in Daucus.
Data from: Degenerate adaptor sequences for detecting PCR duplicates in reduced representation sequencing data improve genotype calling accuracy
RAD-tag is a powerful tool for high-throughput genotyping. It relies on PCR amplification of the starting material, following enzymatic digestion and sequencing adaptor ligation. Amplification introduces duplicate reads into the data, which arise from the same template molecule and are statistically nonindependent, potentially introducing errors into genotype calling. In shotgun sequencing, data duplicates are removed by filtering reads starting at the same position in the alignment. However, restriction enzymes target specific locations within the genome, causing reads to start in the same place, and making it difficult to estimate the extent of PCR duplication. Here, we introduce a slight change to the Illumina sequencing adaptor chemistry, appending a unique four-base tag to the first index read, which allows duplicate discrimination in aligned data. This approach was validated on the Illumina MiSeq platform, using double-digest libraries of ants (Wasmannia auropunctata) and yeast (Saccharomyces cerevisiae) with known genotypes, producing modest though statistically significant gains in the odds of calling a genotype accurately. More importantly, removing duplicates also corrected for strong sample-to-sample variability of genotype calling accuracy seen in the ant samples. For libraries prepared from low-input degraded museum bird samples (Mixornis gularis), which had low complexity, having been generated from relatively few starting molecules, adaptor tags show that virtually all of the genotypes were called with inflated confidence as a result of PCR duplicates. Quantification of library complexity by adaptor tagging does not significantly increase the difficulty of the overall workflow or its cost, but corrects for differences in quality between samples and permits analysis of low-input material.
Genotyping by sequencing data of five legume tree species widespread in the rainforests of West and Central Africa
<p>Although today the forest cover is continuous in Central Africa this may have not always been the case, as the scarce fossil record in this region suggests that arid conditions might have significantly reduced tree density during the Ice Ages. Our aim was to investigate whether the dry ice-age periods left a genetic signature on tree species that can be used to infer the date of the past fragmentation of the rainforest. We sequenced reduced representation libraries of 182 samples representing five widespread Legume trees and seven outgroups. Phylogenetic analyses identified an early divergent lineage for all species in West Africa (Upper Guinea), and two clades in Central Africa: Lower Guinea-North and Lower Guinea-South. As the structure separating the Northern and Southern clades -congruent across species- cannot be explained by geographic barriers, we tested other hypotheses with demographic model testing using ∂a∂I. The best estimates indicate that the two clades split between the Upper Pliocene and the Pleistocene, a date compatible with forest fragmentation driven by ice-age climatic oscillations. Furthermore, we found remarkably older split dates for the shade-tolerant tree species with non-assisted seed dispersal than for light-demanding species with long-distance wind dispersal. Different recolonisation abilities after recurrent cycles of forest fragmentation seem to explain why species with long-distance dispersal show more recent genetic admixture between the two clades than species with limited seed dispersal. Despite their old history, our results depict the African rainforests as a dynamic biome where tree species have expanded relatively recently after the last glaciation.</p>
Data from: GIbPSs: a toolkit for fast and accurate analyses of genotyping-by-sequencing data without a reference genome
Genotyping-by-sequencing (GBS) and related methods are increasingly used for studies of non-model organisms from population genetic to phylogenetic scales. We present GIbPSs, a new genotyping toolkit for the analysis of data from various protocols such as RAD, double-digest RAD, GBS, and two-enzyme GBS without a reference genome. GIbPSs can handle paired-end GBS data and is able to assign reads from both strands of a restriction fragment to the same locus. GIbPSs is most suitable for population genetic and phylogeographic analyses. It avoids genotyping errors due to indel variation by identifying and discarding affected loci. GIbPSs creates a genotype database that offers rich functionality for data filtering and export in numerous formats. We performed comparative analyses of simulated and real GBS data with GIbPSs and another program, pyRAD. This program accounts for indel variation by aligning homologous sequences. GIbPSs performed better than pyRAD in several aspects. It required much less computation time and displayed higher genotyping accuracy. GIbPSs retained smaller numbers of loci overall in analyses of real GBS data. It nevertheless delivered more complete genotype matrices with greater locus overlap between individuals and greater numbers of loci sampled in all individuals.
Data from: Large-scale genotyping of highly polymorphic loci by next generation sequencing: how to overcome the challenges to reliably genotype individuals?
Studying the different roles of adaptive genes is still a challenge in evolutionary ecology and requires reliable genotyping of large numbers of individuals. Next-generation sequencing (NGS) techniques enable such large-scale sequencing, but stringent data processing is required. Here, we develop an easy to use methodology to process amplicon-based NGS data and we apply this methodology to reliably genotype four major histocompatibility complex (MHC) loci belonging to MHC class I and II of Alpine marmots (Marmota marmota). Our post-processing methodology allowed us to increase the number of retained reads. The quality of genotype assignment was further assessed using three independent validation procedures. A total of 3069 high-quality MHC genotypes were obtained at four MHC loci for 863 Alpine marmots with a genotype assignment error rate estimated as 0.21%. The proposed methodology could be applied to any genetic system and any organism, except when extensive copy-number variation occurs (that is, genes with a variable number of copies in the genotype of an individual). Our results highlight the potential of amplicon-based NGS techniques combined with adequate post-processing to obtain the large-scale highly reliable genotypes needed to understand the evolution of highly polymorphic functional genes.
Data from: Development of highly reliable in silico SNP resource and genotyping assay from exome capture and sequencing: an example from black spruce (Picea mariana)
Picea mariana is a widely distributed boreal conifer across Canada and the subject of advanced breeding programs for which population genomics and genomic selection approaches are being developed. Targeted sequencing was achieved after capturing P. mariana exome with probes designed from the sequenced transcriptome of Picea glauca, a distant relative. A high capture efficiency of 75.9% was reached although spruce has a complex and large genome including gene sequences interspersed by some long introns. The results confirmed the relevance of using probes from congeneric species to perform successfully interspecific exome capture in the genus Picea. A bioinformatics pipeline was developed including stringent criteria that helped detect a set of 97 075 highly reliable in silico SNPs. These SNPs were distributed across 14 909 genes. Part of an Infinium iSelect array was used to estimate the rate of true positives by validating 4267 of the predicted in silico SNPs by genotyping trees from P. mariana populations. The true positive rate was 96.2%, for in silico SNPs compared to a genotyping success rate of 96.7% for a set 1115 P. mariana control SNPs recycled from previous genotyping arrays. These results indicate the high success rate of the genotyping array and the relevance of the selection criteria used to delineate the new P. mariana in silico SNP resource. Furthermore, in silico SNPs were generally of medium to high frequency in natural populations, thus providing high informative value for future population genomics applications.
Data from: Identification of multiple QTL hotspots in sockeye salmon (Oncorhynchus nerka) using genotyping-by-sequencing and a dense linkage map
Understanding the genetic architecture of phenotypic traits can provide important information about the mechanisms and genomic regions involved in local adaptation and speciation. Here, we used genotyping-by-sequencing and a combination of previously published and newly generated data to construct sex-specific linkage maps for sockeye salmon (Oncorhynchus nerka). We then used the denser female linkage map to conduct quantitative trait locus (QTL) analysis for 4 phenotypic traits in 3 families. The female linkage map consisted of 6322 loci distributed across 29 linkage groups and was 4082 cM long, and the male map contained 2179 loci found on 28 linkage groups and was 2291 cM long. We found 26 QTL: 6 for thermotolerance, 5 for length, 9 for weight, and 6 for condition factor. QTL were distributed nonrandomly across the genome and were often found in hotspots containing multiple QTL for a variety of phenotypic traits. These hotspots may represent adaptively important regions and are excellent candidates for future research. Comparing our results with studies in other salmonids revealed several regions with overlapping QTL for the same phenotypic trait, indicating these regions may be adaptively important across multiple species. Altogether, our study demonstrates the utility of genomic data for investigating the genetic basis of important phenotypic traits. Additionally, the linkage map created here will enable future research on the genetic basis of phenotypic traits in salmon.
Data from: Genome assembly improvement and mapping convergently evolved skeletal traits in sticklebacks with genotyping-by-sequencing
Marine populations of the threespine stickleback (Gasterosteus aculeatus) have repeatedly colonized and rapidly adapted to freshwater habitats, providing a powerful system to map the genetic architecture of evolved traits. Here, we developed and applied a binned genotyping-by-sequencing (GBS) method to build dense genome-wide linkage maps of sticklebacks using two large marine by freshwater F2 crosses of more than 350 fish each. The resulting linkage maps significantly improve the genome assembly by anchoring 78 new scaffolds to chromosomes, reorienting 40 scaffolds, and rearranging scaffolds in 4 locations. In the revised genome assembly, 94.6% of the assembly was anchored to a chromosome. To assess linkage map quality, we mapped quantitative trait loci (QTL) controlling lateral plate number, which mapped as expected to a 200-kb genomic region containing Ectodysplasin, as well as a chromosome 7 QTL overlapping a previously identified modifier QTL. Finally, we mapped eight QTL controlling convergently evolved reductions in gill raker length in the two crosses, which revealed that this classic adaptive trait has a surprisingly modular and nonparallel genetic basis.
FIGURES 1‒3 in A revision of the genus Lepidobrya Womersley (Collembola: Entomobryidae) based on morphology and sequence data of the genotype
FIGURES 1‒3. Colour pattern of two individuals in Lepidobrya mawsoni. Scale bars: 1 mm.
Data from: Genotyping-in-Thousands by sequencing of archival fish scales reveals maintenance of genetic variation following a severe demographic contraction in kokanee salmon
<p>Historical DNA analysis of archival samples has added new dimensions to population genetic studies, enabling spatiotemporal approaches for reconstructing population histories and informing conservation management. Here we tested the efficacy of Genotyping-in-Thousands by sequencing (GT-seq) for collecting targeted single nucleotide polymorphism (SNP) genotypic data from archival scale samples, and demonstrate its application to a study of kokanee salmon (<i>Oncorhynchus nerka</i>) in Kluane National Park and Reserve (KNPR; Yukon, Canada) that underwent a severe 12-year population decline followed by a rapid rebound. We genotyped archival scales sampled pre-crash and contemporary fin clips collected post-crash, revealing high coverage (>90% average genotyping across all individuals) and low genotyping error (<0.01% within-libraries, 0.60% among-libraries) despite the relatively poor quality of recovered DNA. We observed slight decreases in expected heterozygosity, allelic diversity, and effective population size post-crash, but none were significant, suggesting genetic diversity was retained despite the severe demographic contraction. Genotypic data also revealed the genetic distinctiveness of a now extirpated population just outside of KNPR, revealing biodiversity loss at the northern edge of the species distribution. More broadly, we demonstrate GT-seq as a valuable tool for genome-wide data collection from archival samples to address basic questions in ecology and evolution, and inform applied research in wildlife conservation and fisheries management.</p>
Data from: Genotyping-in-Thousands by Sequencing panel development and application for high-resolution monitoring of introgressive hybridization within sockeye salmon
<p>Stocking programs have been widely implemented to re-establish extirpated fish species to their historical ranges; when employed in species with complex life histories, such management activities should include careful consideration of resulting hybridization dynamics with resident stocks and corresponding outcomes on recovery initiatives. Genetic monitoring can be instrumental for quantifying the extent of introgression over time, however, conventional markers typically have limited power for the identification of advanced hybrid classes, especially at the intra-specific level. Here, we demonstrate a workflow for developing, evaluating, and deploying a Genotyping-in-Thousands by Sequencing (GT-seq) SNP panel with the power to detect advanced hybrid classes to assess the extent and trajectory of intra-specific hybridization, using the sockeye salmon (<em>Oncorhynchus nerka)</em> stocking program in Skaha Lake, British Columbia, as a case study. Previous analyses detected significant levels of hybridization between the anadromous (sockeye) and freshwater resident (kokanee) forms of <em>O. nerka</em>, but were restricted to assigning individuals to pure-stock or "hybrid". Simulation analyses indicated our GT-seq panel had high accuracy, efficiency and power (> 94.5%) of assignment to pure-stock sockeye salmon/kokanee, F<sub>1</sub>, F<sub>2</sub>, and B<sub>2</sub> backcross-sockeye/kokanee. Re-analysis of 2016/2017 spawners previously analyzed using TaqMan<span> </span>assays and otolith microchemistry revealed shifts in assignment of some hybrids to adjacent pure-stock or B<sub>2</sub>-backcross classes, while new assignment of 2019 spawners revealed hybrids comprised 31% of the population, ~74% of which were B<sub>2</sub>-backcross or F<sub>2</sub>. Overall, the GT-seq panel development workflow presented here could be applied to virtually any system where genetic stock identification and intra-specific hybridization are important management parameters.</p>
Genotype by sequencing data from CHPRRU2, a panel of 282 maize inbred lines.
<p>Genotype by sequencing data from CHPRRU2, a panel of 282 maize inbred lines.</p>
Data from: Paralogs are revealed by proportion of heterozygotes and deviations in read ratios in genotyping by sequencing data from natural populations
Whole genome duplications have occurred in the recent ancestors of many plants, fish, and amphibians, resulting in a pervasiveness of paralogous loci and the potential for both disomic and tetrasomic inheritance in the same genome. Paralogs can be difficult to reliably genotype and are often excluded from genotyping-by-sequencing (GBS) analyses; however, removal requires paralogs to be identified which is difficult without a reference genome. We present a method for identifying paralogs in natural populations by combining two properties of duplicated loci: 1) the expected frequency of heterozygotes exceeds that for singleton loci, and 2) within heterozygotes, observed read ratios for each allele in GBS data will deviate from the 1:1 expected for singleton (diploid) loci. These deviations are often not apparent within individuals, particularly when sequence coverage is low; but, we postulated that summing allele reads for each locus over all heterozygous individuals in a population would provide sufficient power to detect deviations at those loci. We identified paralogous loci in three species: Chinook salmon (Oncorhynchus tshawytscha) which retains regions with ongoing residual tetrasomy on eight chromosome arms following a recent whole genome duplication, mountain barberry (Berberis alpina) which has a large proportion of paralogs that arose through an unknown mechanism, and dusky parrotfish (Scarus niger) which has largely re-diploidized following an ancient whole genome duplication. Importantly, this approach only requires the genotype and allele-specific read counts for each individual, information which is readily obtained from most GBS analysis pipelines.
Data from: Genotyping-by-sequencing provides the first well-resolved phylogeny for coffee (Coffea) and insights into the evolution of caffeine content in its species: GBS coffee phylogeny and the evolution of caffeine content
A comprehensive and meaningful phylogenetic hypothesis for the commercially important coffee genus (Coffea) has long been a key objective for coffee researchers. For molecular studies, progress has been limited by low levels of sequence divergence, leading to insufficient topological resolution and statistical support in phylogenetic trees, particularly for the major lineages and for the numerous species occurring in Madagascar. We report here the first almost fully resolved, broadly sampled phylogenetic hypothesis for coffee, the result of combining genotyping-by-sequencing (GBS) technology with a newly developed, lab-based workflow to integrate short read next-generation sequencing for low numbers of additional samples. Biogeographic patterns indicate either Africa or Asia (or possibly the Arabian Peninsula) as the most likely ancestral locality for the origin of the coffee genus, with independent radiations across Africa, Asia, and the Western Indian Ocean Islands (including Madagascar and Mauritius). The evolution of caffeine, an important trait for commerce and society, was evaluated in light of our phylogeny. High and consistent caffeine content is found only in species from the equatorial, fully humid environments of West and Central Africa, possibly as an adaptive response to increased levels of pest predation. Moderate caffeine production, however, evolved at least one additional time recently (between 2 and 4 Mya) in a Madagascan lineage, which suggests that either the biosynthetic pathway was already in place during the early evolutionary history of coffee, or that caffeine synthesis within the genus is subject to convergent evolution, as is also the case for caffeine synthesis in coffee versus tea and chocolate.
Genotype by sequencing SNP dataset for Malus domestica x Malus sieversii F1 populations GMAL4591 and GMAL4592
<p>Fire blight, a bacterial disease caused by <em>Erwinia</em> <em>amylovora</em>, is the most devastating disease of apples and a major threat to apple production. Most commercial apple cultivars are susceptible to fire blight driving the need to develop fire blight resistant cultivars. Although several major fire blight resistance QTLs have been identified from wild species of <em>Malus</em>, the challenges of breeding apples due to long juvenile phase and heterozygosity greatly limit their use. <em>M. sieversii</em>, the primary progenitor of domesticated apples, is one of the wild <em>Malus</em> species that is sexually compatible with <em>M. domestica</em> and has some favorable fruit quality traits. In this study, we performed QTL analysis on two F1 apple populations of <em>M. domestica</em> cv. 'Royal Gala' × <em>M. sieversi</em>i (GMAL4591 and GMAL4592) to identify fire blight resistance QTL. Parental linkage maps were constructed for each family using marker sets of approximately 20K GBS-SNPs. Phenotype data was collected from parents and progeny through controlled fire blight inoculations in the greenhouse for two subsequent years. A significant (P < 0.0001) moderate-effect fire blight resistance QTL on linkage group 7 of GMAL4591 was identified from the paternal parent<em> M. sieversii</em> 'KAZ 95 17-14' (<em>Msv_FB7</em>). <em>Msv_FB7</em> explains about 48–53% of the phenotyping variance across multiple years and time points. Additionally, a significant (P < 0.001) minor effect QTL explaining 18% of the phenotypic variance was identified in population GMAL4592 on LG10 from 'Royal Gala'. We developed diagnostic SSR markers flanking the Msv_FB7 QTL to use in apple breeding. These findings have the potential to accelerate the development of fire-blight-resistant cultivars.</p>
Method Comparison Study of the Spartan FRX CYP2C19 Genotyping System Against Bi-directional Sequencing
ClinicalTrials.gov study NCT01718535. IPD Sharing: Not stated. Countries: 1. Publications: 0.
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