• Title/Summary/Keyword: Genotyping By Sequencing

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Recent Advances in DNA Sequencing by End-labeled Free-Solution Electrophoresis (ELFSE)

  • Won, Jong-In
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.3
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    • pp.179-186
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    • 2006
  • End-Labeled Free-Solution Electrophoresis (ELFSE) is a new technique that is a promising bioconjugate method for DNA sequencing (or separation) and genotyping by both capillary and microfluidic device electrophoresis. Because ELFSE enables high-resolution electrophoretic separation in aqueous buffer alone (i.e., without a polymer matrix), it eliminates the need to load viscous polymer networks into electrophoresis microchannels. To achieve microchannel DNA separations with high performance, ELFSE requires monodisperse perturbing entities (i.e., drag-tags), which create a large amount of frictional drag when pulled behind DNA during free-solution electrophoresis, and which have other properties suitable for microchannel electrophoresis. In this article, the theoretical concepts of ELFSE and the required characteristics of the drag-tag molecules for the ultimate performance of ELFSE are reviewed. Additionally, the merits and limitations of current drag-tags are also discussed in the context of recent experimental data of ELFSE separation (or sequencing).

Construction of Genetic Linkage Map and Identification of Quantitative Trait Loci in Populus davidiana using Genotyping-by-sequencing (Genotyping-by-sequencing 기법을 이용한 사시나무(Populus davidiana) 유전연관지도 작성 및 양적형질 유전자좌 탐색)

  • Suvi Kim;Yang-gil Kim;Dayoung Lee;Hye-jin Lee;Kyu-Suk Kang
    • Journal of Korean Society of Forest Science
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    • v.112 no.1
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    • pp.40-56
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    • 2023
  • Tree species within the Populus genus grow rapidly and have an excellent capacity to absorb carbon, conferring substantial ability to effective purify the environment. Poplar breeding can be achieved rapidly and efficiently if a genetic linkage map is constructed and quantitative trait loci (QTLs) are identified. Here, a high-density genetic linkage map was constructed for the control pollinated progeny using the genotyping-by-sequencing (GBS) technique, which is a next-generation sequencing method. A search was also performed for the genes associated with quantitative traits located in the genetic linkage map by examining the variables of height and diameter at root collar, and resilience to insect damage. The height and diameter at root collar were measured directly, while the ability to recover from insect damage was scored in a 4-year-old breeding population of aspen hybrids (Odae19 × Bonghyeon4 F1) established in the research forest of Seoul National University. After DNA extraction, paternity was confirmed using five microsatellite markers, and only the individuals for which paternity was confirmed were used for the analysis. The DNA was cut using restriction enzymes and the obtained DNA fragments were prepared using a GBS library and sequenced. The analyzed results were sorted using Populus trichocarpa as a reference genome. Overall, 58,040 aligned single-nucleotide polymorphism (SNP) markers were identified, 17,755 of which were used for mapping genetic linkages. The genetic linkage map was divided into 19 linkage groups, with a total length of 2,129.54 cM. The analysis failed to identify any growth-related QTLs, but a gene assumed to be related to recovery from insect damage was identified on linkage group (chromosome) 4 through genome-wide association study.

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

  • Quan, Juan-Hua;Kim, Tae-Yun;Choi, In-Uk;Lee, Young-Ha
    • Parasites, Hosts and Diseases
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    • v.46 no.2
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    • pp.105-108
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    • 2008
  • Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.

Information Technology Infrastructure for Agriculture Genotyping Studies

  • Pardamean, Bens;Baurley, James W.;Perbangsa, Anzaludin S.;Utami, Dwinita;Rijzaani, Habib;Satyawan, Dani
    • Journal of Information Processing Systems
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    • v.14 no.3
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    • pp.655-665
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    • 2018
  • In efforts to increase its agricultural productivity, the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development has conducted a variety of genomic studies using high-throughput DNA genotyping and sequencing. The large quantity of data (big data) produced by these biotechnologies require high performance data management system to store, backup, and secure data. Additionally, these genetic studies are computationally demanding, requiring high performance processors and memory for data processing and analysis. Reliable network connectivity with large bandwidth to transfer data is essential as well as database applications and statistical tools that include cleaning, quality control, querying based on specific criteria, and exporting to various formats that are important for generating high yield varieties of crops and improving future agricultural strategies. This manuscript presents a reliable, secure, and scalable information technology infrastructure tailored to Indonesian agriculture genotyping studies.

New Lung Cancer Panel for High-Throughput Targeted Resequencing

  • Kim, Eun-Hye;Lee, Sunghoon;Park, Jongsun;Lee, Kyusang;Bhak, Jong;Kim, Byung Chul
    • Genomics & Informatics
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    • v.12 no.2
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    • pp.50-57
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    • 2014
  • We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at $30{\times}$ sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than $1,000{\times}$ coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening.

Improvement of SNPs detection efficient by reuse of sequences in Genotyping By Sequencing technology (유전체 서열 재사용을 이용한 Genotyping By Sequencing 기술의 단일 염기 다형성 탐지 효율 개선)

  • Baek, Jeong-Ho;Kim, Do-Wan;Kim, Junah;Lee, Tae-Ho
    • Journal of the Korea Institute of Information and Communication Engineering
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    • v.19 no.10
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    • pp.2491-2499
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    • 2015
  • Recently, the most popular technique to determine the Genotype, genetic features of individual organisms, is the GBS based on SNP from sequences determined by NGS. As analyzing the sequences by the GBS, TASSEL is the most used program to identify the genotypes. But, TASSEL has limitation that it uses only the partial sequences that is obtained by NGS. We tried to improve the efficiency in use of the sequences in order to solve the limitation. So, we constructed new data sets by quality checking, filtering the unused sequences with error rate below 0.1% and clipping the sequences considering the location of barcode and enzyme. As a result, approximately over 17% of the SNP detection efficiency was increased. In this paper, we suggest the method and the applied programs in order to detect more SNPs by using the disused sequences.

Current Pharmacogenetics in Psychiatry (정신의학에서의 약물유전학 현황)

  • Kim, Il Bin;Lee, Yu Sang
    • Korean Journal of Biological Psychiatry
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    • v.28 no.1
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    • pp.1-6
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    • 2021
  • Pharmacogenetics is opening a new era of precision medicine in psychiatry. Drug-metabolizing enzymes are characterized by genetic polymorphisms, which render a large portion of variability in individual drug metabolism. Dose adjustment based on pharmacogenetics knowledge is a first step to translate pharmacogenetics into clinical practice. However, diverse factors including cost-effectiveness should be addressed to provide clinical recommendation. To address current challenges in pharmacogenetics testing in psychiatry, this review provides an update regarding genotyping (SNP analysis, array, and next-generation sequencing), genotype-phenotype correlations, and cost-effectiveness. The current updates on pharmacogenetics in psychiatry will provide guidance for both clinician and researchers to have a consensus in harmonizing efforts to advance the pharmacogenetics field in a part of precision medicine in psychiatry.

Development of an SNP set for marker-assisted breeding based on the genotyping-by-sequencing of elite inbred lines in watermelon (수박 엘리트 계통의 GBS를 통한 마커이용 육종용 SNP 마커 개발)

  • Lee, Junewoo;Son, Beunggu;Choi, Youngwhan;Kang, Jumsoon;Lee, Youngjae;Je, Byoung Il;Park, Younghoon
    • Journal of Plant Biotechnology
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    • v.45 no.3
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    • pp.242-249
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    • 2018
  • This study was conducted to develop an SNP set that can be useful for marker-assisted breeding (MAB) in watermelon (Citrullus. lanatus L) using Genotyping-by-sequencing (GBS) analysis of 20 commercial elite watermelon inbreds. The result of GBS showed that 77% of approximately 1.1 billion raw reads were mapped on the watermelon genome with an average mapping region of about 4,000 Kb, which indicated genome coverage of 2.3%. After the filtering process, a total of 2,670 SNPs with an average depth of 31.57 and the PIC (Polymorphic Information Content) value of 0.1~0.38 for 20 elite inbreds were obtained. Among those SNPs, 55 SNPs (5 SNPs per chromosome that are equally distributed on each chromosome) were selected. For the understanding genetic relationship of 20 elite inbreds, PCA (Principal Component Analysis) was carried out with 55 SNPs, which resulted in the classification of inbreds into 4 groups based on PC1 (52%) and PC2 (11%), thus causing differentiation between the inbreds. A similar classification pattern for PCA was observed from hierarchical clustering analysis. The SNP set developed in this study has the potential for application to cultivar identification, F1 seed purity test, and marker-assisted backcross (MABC) not only for 20 elite inbreds but also for diverse resources for watermelon breeding.

Genomic Tools and Their Implications for Vegetable Breeding

  • Phan, Ngan Thi;Sim, Sung-Chur
    • Horticultural Science & Technology
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    • v.35 no.2
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    • pp.149-164
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    • 2017
  • Next generation sequencing (NGS) technologies have led to the rapid accumulation of genome sequences through whole-genome sequencing and re-sequencing of crop species. Genomic resources provide the opportunity for a new revolution in plant breeding by facilitating the dissection of complex traits. Among vegetable crops, reference genomes have been sequenced and assembled for several species in the Solanaceae and Cucurbitaceae families, including tomato, pepper, cucumber, watermelon, and melon. These reference genomes have been leveraged for re-sequencing of diverse germplasm collections to explore genome-wide sequence variations, especially single nucleotide polymorphisms (SNPs). The use of genome-wide SNPs and high-throughput genotyping methods has led to the development of new strategies for dissecting complex quantitative traits, such as genome-wide association study (GWAS). In addition, the use of multi-parent populations, including nested association mapping (NAM) and multiparent advanced generation intercross (MAGIC) populations, has helped increase the accuracy of quantitative trait loci (QTL) detection. Consequently, a number of QTL have been discovered for agronomically important traits, such as disease resistance and fruit traits, with high mapping resolution. The molecular markers for these QTL represent a useful resource for enhancing selection efficiency via marker-assisted selection (MAS) in vegetable breeding programs. In this review, we discuss current genomic resources and marker-trait association analysis to facilitate genome-assisted breeding in vegetable species in the Solanaceae and Cucurbitaceae families.