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

2019 ◽  
Author(s):  
Charlotte E. Eriksson ◽  
Joel Ruprecht ◽  
Taal Levi
Keyword(s):  
Amplicon Sequencing ◽  
Error Rates ◽  
Snp Genotyping ◽  
Individual Identification ◽  
Degraded Dna ◽  
Nucleotide Polymorphisms ◽  
Success Rates ◽  
Noninvasive Methods ◽  
Promising Alternative ◽  
Management Actions

AbstractNon-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. The information lost when genotyping success is low may lead to decreased precision in animal population densities which could misguide conservation and management actions. 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. Here, we outline a detailed protocol for cost-effective and accurate noninvasive SNP genotyping using highly multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis latrans). 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 at a cost of under $5 per PCR replicate (excluding labor). 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.

scholarly journals Comparison of single nucleotide polymorphisms and microsatellites in non-invasive genetic monitoring of a wolf population

2012 ◽  
Vol 64 (1) ◽  
pp. 321-335 ◽  
Author(s):  
Elena Fabbri ◽  
R. Caniglia ◽  
Nadia Mucci ◽  
H.P. Thomsen ◽  
K. Krag ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Error Rates ◽  
Snp Genotyping ◽  
Genetic Monitoring ◽  
Taqman Probe ◽  
Degraded Dna ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Non Invasive ◽  
Probe Assay

Single nucleotide polymorphisms (SNPs) which represent the most widespread source of sequence variation in genomes, are becoming a routine application in several fields such as forensics, ecology and conservation genetics. Their use, requiring short amplifications, may allow a more efficient genotyping of degraded DNA. We provide the first application of SNP genotyping in an Italian non-invasive genetic monitoring project of the wolf. We compared three different techniques for genotyping SNPs: pyrosequencing, SNaPshot? and TaqMan? Probe Assay in Real-Time PCR. We successively genotyped nine SNPs using the TaqMan Probe Assay in 51 Italian wolves, 57 domestic dogs, 15 wolf x dog hybrids and 313 wolf scats collected in the northern Apennines. The obtained results were used to estimate genetic variability and PCR error rates in SNP genotyping protocols compared to standard microsatellite analysis. We evaluated the cost, laboratory effort and reliability of these different markers and discuss the possible future use of VeraCode, SNPlex and Fluidigm EP1 system in wild population monitoring.

scholarly journals Sample identification and pedigree reconstruction in Wolverine (Gulo gulo) using SNP genotyping of non-invasive samples

2021 ◽  
Author(s):  
Robert Ekblom ◽  
Malin Aronsson ◽  
Franziska Elsner-Gearing ◽  
Malin Johansson ◽  
Toby Fountain ◽  
...  
Keyword(s):  
Snp Array ◽  
Snp Genotyping ◽  
Individual Identification ◽  
Nucleotide Polymorphisms ◽  
Genetic Management ◽  
Gulo Gulo ◽  
Non Invasive ◽  
Species Of Conservation Concern ◽  
Different Types ◽  
Improved Performance

AbstractFor conservation genetic studies using non-invasively collected samples, genome-wide data may be hard to acquire. Until now, such studies have instead mostly relied on analyses of traditional genetic markers such as microsatellites (SSRs). Recently, high throughput genotyping of single nucleotide polymorphisms (SNPs) has become available, expanding the use of genomic methods to include non-model species of conservation concern. We have developed a 96-marker SNP array for use in applied conservation monitoring of the Scandinavian wolverine (Gulo gulo) population. By genotyping more than a thousand non-invasively collected samples, we were able to obtain precise estimates of different types of genotyping errors and sample dropout rates. The SNP panel significantly outperforms the SSR markers (and DBY intron markers for sexing) both in terms of precision in genotyping, sex assignment and individual identification, as well as in the proportion of samples successfully genotyped. Furthermore, SNP genotyping offers a simplified laboratory and analysis pipeline with fewer samples needed to be repeatedly genotyped in order to obtain reliable consensus data. In addition, we utilised a unique opportunity to successfully demonstrate the application of SNP genotype data for reconstructing pedigrees in wild populations, by validating the method with samples from wild individuals with known relatedness. By offering a simplified workflow with improved performance, we anticipate this methodology will facilitate the use of non-invasive samples to improve genetic management of many different types of populations that have previously been challenging to survey.

scholarly journals Parentage Analysis in Giant Grouper (Epinephelus lanceolatus) Using Microsatellite and SNP Markers from Genotyping-by-Sequencing Data

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1042
Author(s):  
Zhuoying Weng ◽  
Yang Yang ◽  
Xi Wang ◽  
Lina Wu ◽  
Sijie Hua ◽  
...  
Keyword(s):  
Fishery Management ◽  
Genotyping By Sequencing ◽  
Parentage Analysis ◽  
Snp Markers ◽  
Individual Identification ◽  
Pedigree Information ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Polymorphic Snps ◽  
Mixed Family

Pedigree information is necessary for the maintenance of diversity for wild and captive populations. Accurate pedigree is determined by molecular marker-based parentage analysis, which may be influenced by the polymorphism and number of markers, integrity of samples, relatedness of parents, or different analysis programs. Here, we described the first development of 208 single nucleotide polymorphisms (SNPs) and 11 microsatellites for giant grouper (Epinephelus lanceolatus) taking advantage of Genotyping-by-sequencing (GBS), and compared the power of SNPs and microsatellites for parentage and relatedness analysis, based on a mixed family composed of 4 candidate females, 4 candidate males and 289 offspring. CERVUS, PAPA and COLONY were used for mutually verification. We found that SNPs had a better potential for relatedness estimation, exclusion of non-parentage and individual identification than microsatellites, and > 98% accuracy of parentage assignment could be achieved by 100 polymorphic SNPs (MAF cut-off < 0.4) or 10 polymorphic microsatellites (mean Ho = 0.821, mean PIC = 0.651). This study provides a reference for the development of molecular markers for parentage analysis taking advantage of next-generation sequencing, and contributes to the molecular breeding, fishery management and population conservation.

scholarly journals Allele frequencies of single nucleotide polymorphisms of clinically important drug-metabolizing enzymes CYP2C9, CYP2C19, and CYP3A4 in a Thai population

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rattanaporn Sukprasong ◽  
Sumonrat Chuwongwattana ◽  
Napatrupron Koomdee ◽  
Thawinee Jantararoungtong ◽  
Santirhat Prommas ◽  
...  
Keyword(s):  
Snp Genotyping ◽  
Allele Frequencies ◽  
Nucleotide Polymorphisms ◽  
Poor Metabolizer ◽  
Thai Population ◽  
Metabolizing Enzymes ◽  
Single Nucleotide ◽  
Genotyping Assay ◽  
Intermediate Metabolizer ◽  
Important Drug

AbstractPrior knowledge of allele frequencies of cytochrome P450 polymorphisms in a population is crucial for the revision and optimization of existing medication choices and doses. In the current study, the frequency of the CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2C19*6, CYP2C19*17, and CYP3A4 (rs4646437) alleles in a Thai population across different regions of Thailand was examined. Tests for polymorphisms of CYP2C9 and CYP3A4 were performed using TaqMan SNP genotyping assay and CYP2C19 was performed using two different methods; TaqMan SNP genotyping assay and Luminex x Tag V3. The blood samples were collected from 1205 unrelated healthy individuals across different regions within Thailand. Polymorphisms of CYP2C9 and CYP2C19 were transformed into phenotypes, which included normal metabolizer (NM), intermediate metabolizer (IM), poor metabolizer (PM), and rapid metabolizers (RM). The CYP2C9 allele frequencies among the Thai population were 0.08% and 5.27% for the CYP2C9*2 and CYP2C9*3 alleles, respectively. The CYP2C19 allele frequencies among the Thai population were 25.60%, 2.50%, 0.10%, and 1.80% for the CYP2C19*2, CYP2C19*3, CYP2C19*6, and CYP2C19*17 alleles, respectively. The allele frequency of the CYP3A4 (rs4646437) variant allele was 28.50% in the Thai population. The frequency of the CYP2C9*3 allele was significantly lower among the Northern Thai population (P < 0.001). The frequency of the CYP2C19*17 allele was significantly higher in the Southern Thai population (P < 0.001). Our results may provide an understanding of the ethnic differences in drug responses and support for the utilization of pharmacogenomics testing in clinical practice.

High Microsatellite and SNP Genotyping Success Rates Established in a Large Number of Genomic DNA Samples Extracted From Mouth Swabs and Genotypes

2006 ◽  
Vol 9 (4) ◽  
pp. 501-506 ◽  
Author(s):  
Josine L. Min ◽  
Nico Lakenberg ◽  
Margreet Bakker-Verweij ◽  
Eka Suchiman ◽  
Dorret I. Boomsma ◽  
...  
Keyword(s):  
Success Rate ◽  
Genomic Dna ◽  
Snp Genotyping ◽  
High Yield ◽  
Success Rates ◽  
Nucleotide Polymorphism ◽  
High Quality ◽  
Single Nucleotide ◽  
Dna Yield ◽  
Yield And Quality

AbstractIn this article, we present the genomic DNA yield and the microsatellite and single nucleotide polymorphism (SNP) genotyping success rates of genomic DNA extracted from a large number of mouth swab samples. In total, the median yield and quality was determined in 714 individuals and the success rates in 378,480 genotypings of 915 individuals. The median yield of genomic DNA per mouth swab was 4.1 μg (range 0.1–42.2 μg) and was not reduced when mouth swabs were stored for at least 21 months prior to extraction. A maximum of 20 mouth swabs is collected per participant. Mouth swab samples showed in, respectively, 89% for 390 microsatellites and 99% for 24 SNPs a genotyping success rate higher than 75%. A very low success rate of genotyping (0%–10%) was obtained for 3.2% of the 915 mouth swab samples using microsatellite markers. Only 0.005% of the mouth swab samples showed a geno-typing success rate lower than 75% (range 58%–71%) using SNPs. Our results show that mouth swabs can be easily collected, stored by our conditions for months prior to DNA extraction and result in high yield and high-quality DNA appropriate for genotyping with high success rate including whole genome searches using microsatellites or SNPs.

scholarly journals Population structure of eulachon Thaleichthys pacificus from Northern California to Alaska using single nucleotide polymorphisms from direct amplicon sequencing

2020 ◽  
Author(s):  
Ben J. G. Sutherland ◽  
John Candy ◽  
Kayla Mohns ◽  
Olivia Cornies ◽  
Kim Jonsen ◽  
...  
Keyword(s):  
Gulf Of Alaska ◽  
Amplicon Sequencing ◽  
Fraser River ◽  
Northern California ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Southeast Alaska ◽  
Southern Us ◽  
Mixed Stock ◽  
Future Work

ABSTRACTEulachon Thaleichthys pacificus, a culturally and ecologically important anadromous smelt (Family Osmeridae), ranges from Northern California to the southeast Bering Sea. In recent decades, some populations have experienced declines. Here we use a contig-level genome assembly combined with previously published RADseq-derived markers to construct an amplicon panel for eulachon. Using this panel, we develop a filtered genetic baseline of 521 variant loci genotyped in 1,989 individuals from 14 populations ranging from Northern California through Central Alaska. Consistent with prior genetic studies, the strongest separation occurs among three main regions: from Northern California up to and including the Fraser River; north of the Fraser River to southeast Alaska; and within the Gulf of Alaska. Separating the Fraser River from southern US populations, and refining additional substructure within the central coast may be possible in mixed-stock analysis; this will be addressed in future work. The amplicon panel outperformed the previous microsatellite panel, and thus will be used in future mixed-stock analyses of eulachon in order to provide new insights for management and conservation of eulachon.

scholarly journals EVOLUTION OF DNA SEQUENCING METHODS AND PROSPECTS FOR THEIR USE IN FORENSIC DNA EXAMINATION

2020 ◽  
Vol 11 (87) ◽  
Author(s):  
Zhanna Bazyliuk ◽  
Keyword(s):  
Dna Sequencing ◽  
Molecular Genetic ◽  
Genetic Research ◽  
Snp Markers ◽  
Degraded Dna ◽  
Phenotypic Traits ◽  
Nucleotide Polymorphisms ◽  
Biological Origin ◽  
Genetic Traits ◽  
Str Loci

The study of the human genome makes it possible to use genetic information to identify individual traits, diagnosis of diseases and forecasting and prevention of their development, promotes a personal approach when choosing treatment methods; population research, ethnogenesis and evolutionary processes. Introduction of DNA sequencing methods in domestic genetic fingerprinting will contribute to a more informative establishment of human genetic traits. The main purpose of molecular genetic research is to establish the genetic features of missing people, their relatives, to conduct paternity, to identify traces of biological origin and their identification. This article talks about the gradual development of DNA sequencing technology, which is conventionally divided into three types. The first type includes sequencing using capillary electrophoresis and pyrosequencing. The second type is high-throughput pyrosequencing, semiconductor, cyclic ligase, and the use of fluorescently labeled precursors, based on the sequencing of millions of DNA fragments simultaneously. The third stage includes methods that do not require prior sample preparation. These are methods of nanoporous sequencing, sequencing of one molecule, one-molecular sequencing. Today, each of the sequencing methods is aimed at performing different tasks. A number of methods are promising in the field of molecular-genetic examination. In world jurisprudence, sequencing is implemented mainly with the help of devices - Illumina’s, MiSeq FGx, Ion Torrent PGM from ThermoFisher and Ion S5. Research in forensic expertise of single nucleotide polymorphisms (SNP), sequencing of STR-loci and mitochondrial DNA, STR-loci and SNP-markers of the Y chromosome, will provide a high level of information, determination of human phenotypic traits, the possibility of establishing genetic traits from significantly degraded DNA. This article deals with modern problems of identification of human genetic traits and the prospect of introduction of the newest methods of sequencing for their qualitative and complete establishment.

scholarly journals Evaluating the accuracy of variant calling methods using the frequency of parent-offspring genotype mismatch

2021 ◽  
Author(s):  
Russ Jasper ◽  
Tegan Krista McDonald ◽  
Pooja Singh ◽  
Mengmeng Lu ◽  
Clément Rougeux ◽  
...  
Keyword(s):  
Pinus Contorta ◽  
Quantitative Assessment ◽  
Variant Calling ◽  
Error Rates ◽  
Snp Genotyping ◽  
Genotyping Error ◽  
Snp Calling ◽  
Full Sibling ◽  
Model Study ◽  
Offspring Genotype

The use of NGS datasets has increased dramatically over the last decade, however, there have been few systematic analyses quantifying the accuracy of the commonly used variant caller programs. Here we used a familial design consisting of diploid tissue from a single Pinus contorta parent and the maternally derived haploid tissue from 106 full-sibling offspring, where mismatches could only arise due to mutation or bioinformatic error. Given the rarity of mutation, we used the rate of mismatches between parent and offspring genotype calls to infer the SNP genotyping error rates of FreeBayes, HaplotypeCaller, SAMtools, UnifiedGenotyper, and VarScan. With baseline filtering HaplotypeCaller and UnifiedGenotyper yielded one to two orders of magnitude larger numbers of SNPs and error rates, whereas FreeBayes, SAMtools and VarScan yielded lower numbers of SNPs and more modest error rates. To facilitate comparison between variant callers we standardized each SNP set to the same number of SNPs using additional filtering, where UnifiedGenotyper consistently produced the smallest proportion of genotype errors, followed by HaplotypeCaller, VarScan, SAMtools, and FreeBayes. Additionally, we found that error rates were minimized for SNPs called by more than one variant caller. Finally, we evaluated the performance of various commonly used filtering metrics on SNP calling. Our analysis provides a quantitative assessment of the accuracy of five widely used variant calling programs and offers valuable insights into both the choice of variant caller program and the choice of filtering metrics, especially for researchers using non-model study systems.

A fully integrated SNP genotyping system for hereditary hearing-loss detection

Lab on a Chip ◽  
2022 ◽  
Author(s):  
Nan Li ◽  
Yuanyue Zhang ◽  
Minjie Shen ◽  
Youchun Xu
Keyword(s):  
Hearing Loss ◽  
Early Intervention ◽  
Single Nucleotide Polymorphisms ◽  
Snp Genotyping ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Fully Integrated ◽  
Hereditary Hearing Loss

Hereditary hearing loss is one of the most common human neurosensory disorder, and there is a great need for early intervention methods such as genetically screening newborns. Single nucleotide polymorphisms...

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