Next Generation Sequencing (NGS), A Key Tool to open the Personalized Medicine Era

( Sun-il Kwon ) 대한임상검사과학회 2012 대한임상검사과학회지(KJCLS) Vol.44 No.4

Next-Generation Sequencing (NGS) is a term that means post-Sanger sequencing methods with highthroughput sequencing technologies. NGS parallelizes the sequencing process, producing thousands or millions of sequences at once. The latest NGS technologies use even single DNA molecule as a template and measures the DNA sequence directly via measuring electronic signals from the extension or degradation of DNA. NGS is making big impacts on biomedical research, molecular diagnosis and personalized medicine. The hospitals are rapidly adopting the use of NGS to help to patients understand treatment with sequencing data. As NGS equipments are getting smaller and affordable, many hospitals are in the process of setting up NGS platforms. In this review, the progress of NGS technology development and action mechanisms of representative NGS equipments of each generation were discussed. The key technological advances in the commercialized platforms were presented. As NGS platforms are a great concern in the healthcare area, the latest trend in the use of NGS and the prospect of NGS in the future in diagnosis and personalized medicine were also discussed.

Toward Complete Bacterial Genome Sequencing Through the Combined Use of Multiple Next-Generation Sequencing Platforms

( Hae Young Jeong ),( Dae Hee Lee ),( Choong Min Ryu ),( Seung Hwan Park ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.1

PacBio’s long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of secondgeneration, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

차세대 시퀀싱 (NGS), 맞춤 의학 시대를 여는 핵심 도구

권순일 대한임상검사과학회 2012 대한임상검사과학회지(KJCLS) Vol.44 No.4

Next-Generation Sequencing (NGS) is a term that means post-Sanger sequencing methods with highthroughput sequencing technologies. NGS parallelizes the sequencing process, producing thousands or millions of sequences at once. The latest NGS technologies use even single DNA molecule as a template and measures the DNA sequence directly via measuring electronic signals from the extension or degradation of DNA. NGS is making big impacts on biomedical research, molecular diagnosis and personalized medicine. The hospitals are rapidly adopting the use of NGS to help to patients understand treatment with sequencing data. As NGS equipments are getting smaller and affordable, many hospitals are in the process of setting up NGS platforms. In this review, the progress of NGS technology development and action mechanisms of representative NGS equipments of each generation were discussed. The key technological advances in the commercialized platforms were presented. As NGS platforms are a great concern in the healthcare area, the latest trend in the use of NGS and the prospect of NGS in the future in diagnosis and personalized medicine were also discussed. .

Next-generation approaches to the microbial ecology of food fermentations

( Nicholas A. Bokulich ),( David A. Mills ) 생화학분자생물학회(구 한국생화학분자생물학회) 2012 BMB Reports Vol.45 No.7

Food fermentations have enhanced human health since the dawn of time and remain a prevalent means of food processing and preservation. Due to their cultural and nutritional importance, many of these foods have been studied in detail using molecular tools, leading to enhancements in quality and safety. Furthermore, recent advances in high-throughput sequencing technology are revolutionizing the study of food microbial ecology, deepening insight into complex fermentation systems. This review provides insight into novel applications of select molecular techniques, particularly next-generation sequencing technology, for analysis of microbial communities in fermented foods. We present a guideline for integrated molecular analysis of food microbial ecology and a starting point for implementing next-generation analysis of food systems. [BMB Reports 2012;45(7): 377-389]

Genotyping-by-Sequencing: a Promising Tool for Plant Genetics Research and Breeding

정용석,김창수,최상철,전태환 한국원예학회 2017 Horticulture, Environment, and Biotechnology Vol.58 No.5

Plant genetics studies and breeding programs utilize the genetic variation caused by DNA polymorphisms. Molecular makers are used to detect these variations in the DNA. The advent of next-generation sequencing (NGS)technologies has conferred new opportunities for high-throughput genotyping in various plant species. Recent improvementsin high-throughput sequencing have enabled sequences to be used to detect and score single nucleotide polymorphisms(SNPs) by bypassing the time-consuming process of marker development. With NGS, whole-genome sequencing dataand millions of genome-wide SNPs for high-throughput genotyping have become available for a variety of geneticstudies and breeding programs. SNP markers are powerful for analyses of integrated SNP sets in a species, althoughhigh costs hinder the wider use of SNPs. However, genotype-by-sequencing (GBS), a series of genetic analyses thatincludes molecular marker discovery and genotyping using NGS technologies, has opened new possibilities in plantbreeding and plant genetics studies. It offers cost-effective genome-wide scanning and multiplexed sequencing platforms. The GBS method uses restriction enzymes coupled with DNA barcoded adapters to reduce complexity. GBS cansimultaneously perform SNP discovery and genotyping with or without reference genome sequences. Therefore, GBScan be applied to various approaches for plant breeding and plant genetics studies, including linkage maps, genome-wideassociation studies, genomic selection, and genomic diversity studies. These features make GBS an ideal tool for studiesranging from single-gene markers to whole-genome profiling.

Three-Year Experience of an External Proficieny Testing Survey for ­Next-Generation Sequencing-Based Testing for Germline Mutation

성문우,Manjin Kim,Ho-Seob Shin,Sung Im Cho,Sung Sup Park 대한임상검사정도관리협회 2020 Journal of Laboratory Medicine And Quality Assuran Vol.42 No.1

Background: Next-generation sequencing is a powerful technology that allows simultaneous analysis of several genes but also demands a well-designed quality management system owing to its complexity. We aimed to analyze the results of next-generation sequencing (NGS)-germline proficiency testing (PT) survey performed by the Korean Association of External Quality Assessment Service during 2017–2019 to assess the current status of the NGS-based genetic testing in Korea.Methods: The recent 3-year results from the PT survey were investigated. During this period, PT survey was performed twice every year with two or three challenges per round. Correct results (%) were calculated from all tested regions; the trend by year and variation type was analyzed and the probable causes estimated.Results: During this period, the number of participating laboratories increased from 5 to 22. The correct results were 97.2% in average and showed a gradual increase with year. The most common ‘unacceptable’ results were false-negative or false-positive, followed by inappropriate nomenclature and zygosity assignment.Conclusions: The PT survey shows that the overall performance of NGS laboratories in Korea is highly confident, although some improvements may be needed. A method-based PT survey for the NGS test serves as a useful approach to assess the performance of NGS laboratories.

차세대 염기서열 분석법을 이용한 15개 상염색체 STR의 염기서열 생성 및 유전자형 분석

김은혜,양인석,정상은,신경진,양우익 대한법의학회 2014 대한법의학회지 Vol.38 No.2

Recently, next generation sequencing (NGS) has received attention as the ultimategenotyping method to overcome the limitations of capillary electrophoresis (CE)-based short tandem repeat (STR) analysis, such as the limited number of STR locithat can be measured simultaneously using fluorescent-labeled primers and the maximumsize of STR amplicons. In this study, we analyzed 15 autosomal STR markersvia the NGS method and evaluated their effectiveness in STR analysis. Using maleand female standard DNA as single-sources and their 1:1 mixture, we sequentiallygenerated sample amplicons by the multiplex polymerase chain reaction (PCR)method, constructed DNA libraries by ligation of adapters with a multiplex identifier(MID), and sequenced DNA using the Roche GS Junior Platform. Sequencing datafor each sample were analyzed via alignment with pre-built reference sequences. Most STR alleles could be determined by applying a coverage threshold of 20% forthe two single-sources and 10% for the 1:1 mixture. The structure of the STR in eachallele was accurately determined by examining the sequences of the target STRregion. The mixture ratio of the mixed sample was estimated by analyzing the coverageratios between assigned alleles at each locus and the reference/variant ratiosfrom the observed sequence variations. In conclusion, the experimental method usedin this study allowed the successful generation of NGS data. In addition, the NGS dataanalysis protocol enables accurate STR allele call and repeat structure determinationat each locus. Therefore, this approach using the NGS system will be helpful to interpretand analysis the STR profiles from singe-source and even mixed samples inforensic investigation.

Application of Next Generation Sequencing in Laboratory Medicine

Zhong Yiming,Xu Feng,Wu Jinhua,Schubert Jeffrey,Li Marilyn M. 대한진단검사의학회 2021 Annals of Laboratory Medicine Vol.41 No.1

The rapid development of next-generation sequencing (NGS) technology, including advances in sequencing chemistry, sequencing technologies, bioinformatics, and data interpretation, has facilitated its wide clinical application in precision medicine. This review describes current sequencing technologies, including short- and long-read sequencing technologies, and highlights the clinical application of NGS in inherited diseases, oncology, and infectious diseases. We review NGS approaches and clinical diagnosis for constitutional disorders; summarize the application of U.S. Food and Drug Administration-approved NGS panels, cancer biomarkers, minimal residual disease, and liquid biopsy in clinical oncology; and consider epidemiological surveillance, identification of pathogens, and the importance of host microbiome in infectious diseases. Finally, we discuss the challenges and future perspectives of clinical NGS tests.

Assessment of the Dynamics of Microbial Community Associated with Tetraselmis suecica Culture under Different LED Lights Using Next-Generation Sequencing

( Su-jeong Yang ),( Hyun-woo Kim ),( Seok-gwan Choi ),( Sangdeok Chung ),( Seok Jin Oh ),( Shweta Borkar ),( Hak Jun Kim ) 한국미생물 · 생명공학회 2019 Journal of microbiology and biotechnology Vol.29 No.12

Tetraselmis is a green algal genus, some of whose species are important in aquaculture as well as biotechnology. In algal culture, fluorescent lamps, traditional light source for culturing algae, are now being replaced by a cost-effective light-emitting diodes (LEDs). In this study, we investigated the effect of LED light of different wavelengths (white, red, yellow, and blue) on the growth of Tetraselmis suecica and its associated microbial community structures using the next-generation sequencing (NGS). The fastest growth rate of T. suecica was shown in the red light, whereas the slowest was in yellow. The highest OTUs (3426) were identified on day 0, whereas the lowest ones (308) were found on day 15 under red light. The top 100 OTUs associated with day 0 and day 5 cultures of T. suecica under the red and yellow LED were compared. Only 26 OTUs were commonly identified among four samples. The highest numbers of unique OTUs were identified at day 0, indicating the high degree of initial microbial diversity of the T. suecica inoculum. The red light-unique OTUs occupied 34.98%, whereas the yellow-specific OTUs accounted for only 2.2%. This result suggested a higher degree of interaction in T. suecica culture under the red light, where stronger photosynthesis occurs. Apparently, the microbial community associated with T. suecica related to the oxygen produced by algal photosynthesis. This result may expand our knowledge about the algaebacteria consortia, which would be useful for various biotechnological applications including wastewater treatment, bioremediation, and sustainable aquaculture.

Next Generation Sequencing을 통한 미생물 군집 분석의 축산분야 활용

김민석,백열창,오영균,Kim, Minseok,Baek, Youlchang,Oh, Young Kyoon 한국축산환경학회 2015 한국축산시설환경학회지 Vol.21 No.3

The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.