The optimal standard protocols for whole-genome sequencing of antibiotic-resistant pathogenic bacteria using third-generation sequencing platforms

La Tae-Min,Kim Ji-hoon,Kim Taesoo,Lee Hong-Jae,이윤석,Shin Hyunjin,송용준,Ahn Gyuhee,Hur Won,이중복,Park Seung-Yong,Choi In-Soo,이상원 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.4

Background The emergence of antibiotic-resistant bacterial pathogens in the environment has been increasing, posing a threat to public health. Next-generation sequencing technology, which is both low cost and large scale, was used to identify antibiotic-resistance genes or toxin genes in these pathogens. Short-read sequencing cannot fully reconstruct bacterial chromosomes and plasmids carrying toxin genes and antibiotic-resistance genes because of their location on the insertion sequences or repeat regions. Third-generation sequencing generated long reads that could cover the repetitive and/or insertion sequences, allowing for complete chromosome and plasmid reconstruction. However, the optimal protocols for whole-genome sequencing of antibiotic-resistance pathogenic bacteria, from DNA extraction to genome assembly, are still being researched. Objective To develop a pipeline of optimal methods for whole-genome sequencing of bacteria, we compared three commercial DNA extraction kits (column extraction, magnetic bead extraction, and precipitation extraction), two third-generation sequencing platforms (MinION and PacBio), and three assembly methods (flye, unicycler, and unicycler_hybrid). The assembly results were evaluated based on the number of contigs and the detection of anti-microbial-resistance genes. Results Magnetic bead extraction method generated longer N50 read lengths and greater read length distribution than the other two extraction methods. The Flye assembler in combination with magnetic bead extraction and MinION sequencing provided consistent successful plasmid assembly results and detected all antimicrobial-resistance gene in all datasets. Conclusion DNA extraction, sequencing platform, and assembly methods can all have an impact on the results of bacterial whole-genome sequencing. Our findings could be a practical protocol for researchers who use third-generation sequencing to perform bacterial whole-genome sequencing by consistently resolving small plasmids carrying antibiotic-resistance genes. Background The emergence of antibiotic-resistant bacterial pathogens in the environment has been increasing, posing a threat to public health. Next-generation sequencing technology, which is both low cost and large scale, was used to identify antibiotic-resistance genes or toxin genes in these pathogens. Short-read sequencing cannot fully reconstruct bacterial chromosomes and plasmids carrying toxin genes and antibiotic-resistance genes because of their location on the insertion sequences or repeat regions. Third-generation sequencing generated long reads that could cover the repetitive and/or insertion sequences, allowing for complete chromosome and plasmid reconstruction. However, the optimal protocols for whole-genome sequencing of antibiotic-resistance pathogenic bacteria, from DNA extraction to genome assembly, are still being researched. Objective To develop a pipeline of optimal methods for whole-genome sequencing of bacteria, we compared three commercial DNA extraction kits (column extraction, magnetic bead extraction, and precipitation extraction), two third-generation sequencing platforms (MinION and PacBio), and three assembly methods (flye, unicycler, and unicycler_hybrid). The assembly results were evaluated based on the number of contigs and the detection of anti-microbial-resistance genes. Results Magnetic bead extraction method generated longer N50 read lengths and greater read length distribution than the other two extraction methods. The Flye assembler in combination with magnetic bead extraction and MinION sequencing provided consistent successful plasmid assembly results and detected all antimicrobial-resistance gene in all datasets. Conclusion DNA extraction, sequencing platform, and assembly methods can all have an impact on the results of bacterial whole-genome sequencing. Our findings could be a practical protocol for researchers who use third-generation sequencing to perform bacterial whole-genome sequencing by consistently resolving small plasmids carrying antibiotic-resistance genes.

뇌전증의 유전적 진단

이윤정(Yun Jeong Lee),안주희(Ju Hee Ahn),권순학(Soon hak Kwon),황수경(Su Kyeong Hwang) 대한소아신경학회 2016 대한소아신경학회지 Vol.24 No.4

최근 유전자 검사 기법의 급속한 발전으로 인해 이전까지 특발성 뇌전증으로 분류되었던 환자들에서 많은 유전적 원인이 밝혀지게 되었고, 맞춤 치료로 연결되는 경우도 늘었다. 바이오헬스 산업이 정밀의료에 초점을 맞추게 되면서 차세대 유전자 검사와 관련된 법과 규제들도 많이 완화가 되었고 보험급여가 시행될 날이 머지않은 시점에 있다. 유전자검사는 유전적 원인을 가진 것으로 의심되는 뇌전증 환자 누구에게나 고려될 수 있지만, 항경련제에 잘 반응하지 않는 난치성 뇌전증 환자들에게 시행하는 편이 적합하고 적용될 수 있는 검사 방법에 따라 (1) 단일 유전자 변이에 의한 질환이 의심되는 경우, (2) 다른 신경/정신학적인 질환을 동반하거나 기형이 있는 경우, (3) 임상양상이 한정적이지 않고 관련될 수 있는 유전자가 여러 개인 경우로 환자를 분류할 수 있다. 적용할 수 있는 유전자 검사로는 Sanger sequencing, chromosome microarray, targeted gene panels, whole exome sequencing, whole genome sequencing이 있다. 단일 유전자 변이에 의한 질환이 의심되는 경우는 Sanger sequencing, 다른 신경/정신학적인 질환을 동반하거나 기형이 있는 경우는 chromosome microarray, 임상양상이 한정적이지 않고 관련될 수 있는 유전자가 여러 개인 경우는 targeted gene panels을 우선 고려할 수 있으며, 검사 결과가 음성인 경우는 whole exome sequencing이나 whole genome sequencing에 이르기까지 순차적으로 검사할 수 있다. Targeted gene panels는 Sanger sequencing이나 whole exome sequencing 등에 비해 염기서열 당 분석 비용이 싼 편이며 진단율이 높고 시간을 절약할 수 있는 장점이 있다. 차세대 유전자 검사의 장점에도 불구하고 아직까지는 임상에서 적극적으로 활용하기 위해 넘어야 할 사회적, 윤리적, 경제적 장벽들이 있다. 본 종설에서는 최근 뇌전증 유전학의 변화와 임상 실제에서 활용할 수 있는 유전자 검사 방법을 제시한다. Major advances in genetics allow us to uncover genetic causes of epilepsy and influence therapeutic decisions. As precision medicine becomes a focus for bio-health industry, laws related to next generation sequencing are improving and its application will continue to expand. Although genetic testing can be considered for anyone who is suspected to have a genetic cause, the most suitable indication is drug-resistant epilepsy. Epileptic patients who have genetic testing can be classified into three groups: (1) patients with defined phenotypes suggesting monogenic epilepsy syndrome; (2) patients with neuropsychiatric comorbidities or dysmorphic features; and (3) patients with undefined phenotypes with multiple candidate genes. Applicable genetic testing methods for epilepsy are as follows: Sanger sequencing, chromosome microarray, targeted gene panels, whole exome sequencing, and whole genome sequencing. Sanger sequencing is indicated for patients with defined phenotypes by a single gene mutation. Chromosome microarray is recommended as part of the initial evaluation of unexplained epilepsy, particularly among patients with associated neuropsychiatric comorbidities or dysmorphic features. Next generation sequencing can be informative in patients with undefined phenotypes with multiple candidate genes. Targeted gene panels are a cost-effective alternative to both Sanger sequencing and whole exome sequencing for the genetic diagnosis of epilepsy. There are, however, social, ethical and even financial hurdles to overcome in the transition of next generation sequencing technology into the clinic. This review focuses on recent transformation of epilepsy genetics and suggests a strategy for actual clinical practice.

Whole Exome Sequencing of a Patient with Duchenne Muscular Dystrophy

최봉석(Bong Seok Choi),황수경(Su-Kyeong Hwang) 대한소아신경학회 2014 대한소아신경학회지 Vol.22 No.1

듀센형 근이영양증은 소아에서 가장 흔하고 치명적인 유전성 근육질환으로, 디스트로핀 유전자의 변이로 인해 발생한다. 디스트로핀 유전자의 결실이나 중복에 의해 생기는 경우가 많으므로 MLPA나 aCGH가 진단을 위해 많이 쓰인다. 그러나 환자의 1/3 에서는 변이가 발견되지 않는데, 이럴 경우 direct sequencing을 통해 MLPA나 aCGH에서 확인되지 않는 미세한 결실, 중복, 또는 point mutation이 있는지 확인하게 된다. 그러나 DMD 유전자는 크기가 매우 크기 때문에 direct sequencing을 하기에는 시간과 비용이 엄청나게 소모되며 최종 결과도 확정적이지 않을 가능성이 있다. 우리는 MLPA에서 변이가 발견되지 않은 진행성 근이영양증 환자를 대상으로 whole exome sequencing을 시행하였는데, 디스트로핀 유전자의 frame shift mutation이 발견되었다. 환자의 어머니를 대상으로 시행한 direct sequencing에서도 환자와 같은 변이가 발견되었다. Whole exome sequencing은 MLPA에서 변이가 발견되지 않는 듀센형 근이영양증 환자의 새로운 진단 도구로 훌륭한 역할을 할 수 있을 것으로 생각된다. Duchenne muscular dystrophy (DMD) is the most common and lethal dystrophy in childhood, caused by mutations in the dystrophin (DMD) gene. Multiplex ligation dependent probe amplification (MLPA) or array comparative genome hybridization (aCGH) is widely used as an initial molecular diagnostic tool. If no deletions or duplications are found in MLPA or aCGH, the samples must be subjected to a second test of direct sequencing. Direct sequencing of the DMD gene, however, is timeconsuming, high-cost, and can be inconclusive. Here, we performed whole exome sequencing on a patient with progressive muscle weakness whose MLPA result was negative; the result revealed a rare frame shift mutation. Direct sequencing on the patient's mother showed the same mutation. Whole exome sequencing can be a new diagnostic routine for DMD patients with negative MLPA3

Heterogeneous Spectrum of CFTR Gene Mutations in Korean Patients with Cystic Fibrosis

정혜영,기창석,고원정,안강모,이상일,김정호,고재성,서정기,차승익,이은실,김종원 대한진단검사의학회 2011 Annals of Laboratory Medicine Vol.31 No.3

Background: Cystic fibrosis (CF) is one of the most common hereditary disorders among Caucasians. The most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been well established among Caucasian populations. In Koreans, however, there are very few cases of genetically confirmed CF thus far, and the spectrum of mutations seems quite different from that observed in Caucasians. Methods: In the present study, we describe the cases of 2 Korean CF patients, present sequencing results identifying mutations in their CFTR gene, and summarize the results of CFTR mutational spectrum from previously reported Korean CF patients. The mutations described were identified by performing direct sequencing analysis of the complete coding regions and flanking intronic sequences of the CFTR gene, followed by multiplex ligation-dependent probe amplification (MLPA) analysis in order to detect gene deletions or duplications that could not be identified by a direct sequencing method. Results: Three CFTR mutations were identified in the 2 patients, including p.Q98R, c.2052delA, and c.579+5G>A. In an analysis of 9 Korean CF patients that included the 2 patients presented in this study, p.Q98R mutation was the only recurrently observed mutation with a frequency of 18.8% (3/16 alleles). Furthermore, only one of the mutations (c.3272-26A>G) was found among the 32 common mutations in the screening panel for Caucasians from the Cystic Fibrosis Mutation Database. Conclusions: Sequencing of the entire CFTR gene followed by MLPA analysis, rather than using the targeted sequencing-based screening panel for mutations commonly found in Caucasian populations, is recommended for genetic analysis of Korean CF patients. Background: Cystic fibrosis (CF) is one of the most common hereditary disorders among Caucasians. The most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been well established among Caucasian populations. In Koreans, however, there are very few cases of genetically confirmed CF thus far, and the spectrum of mutations seems quite different from that observed in Caucasians. Methods: In the present study, we describe the cases of 2 Korean CF patients, present sequencing results identifying mutations in their CFTR gene, and summarize the results of CFTR mutational spectrum from previously reported Korean CF patients. The mutations described were identified by performing direct sequencing analysis of the complete coding regions and flanking intronic sequences of the CFTR gene, followed by multiplex ligation-dependent probe amplification (MLPA) analysis in order to detect gene deletions or duplications that could not be identified by a direct sequencing method. Results: Three CFTR mutations were identified in the 2 patients, including p.Q98R, c.2052delA, and c.579+5G>A. In an analysis of 9 Korean CF patients that included the 2 patients presented in this study, p.Q98R mutation was the only recurrently observed mutation with a frequency of 18.8% (3/16 alleles). Furthermore, only one of the mutations (c.3272-26A>G) was found among the 32 common mutations in the screening panel for Caucasians from the Cystic Fibrosis Mutation Database. Conclusions: Sequencing of the entire CFTR gene followed by MLPA analysis, rather than using the targeted sequencing-based screening panel for mutations commonly found in Caucasian populations, is recommended for genetic analysis of Korean CF patients.

A Sequencing Problem with Fuzzy Preference Relation and its Genetic Algorithm-based Solution

이건명(Keon-Myung Lee),손봉기(Bong-Ki Sohn) 한국지능시스템학회 2004 한국지능시스템학회논문지 Vol.14 No.1

A sequencing problem is to find an ordered sequence of some entities which maximizes (or minimize) the domain-specific objective function. As some typical examples of sequencing problems, there are traveling salesman problem, job shop scheduling, flow shop scheduling, and so on. This paper introduces a new type of sequencing problems, named a sequencing problem with fuzzy preference relation, where a fuzzy preference relation is provided for the evaluation of the quality of sequences. It presents how such a problem can be formulated in terms of objective function. It also proposes a genetic algorithm applicable to such a sequencing problem.

Additional Value of Metagenomic Sequencing in Diagnosis of Ventilatorassociated Pneumonia

( Ha Won Hwang ),( Jinwoo Lee ),( Hyoshin Son ),( Chang Kyung Kang ),( Hong Yeul Lee ),( Sang-min Lee ),( Jangsup Moon ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

Background Ventilator associated pneumonia (VAP) is associated with high mortality and morbidity. Unfortunately, conventional Methods often fail to identify the etiology of VAP and accurate microbial diagnosis remains difficult. As a result, broad-spectrum antimicrobial treatment is frequently used for long durations without clear evidence. We compared the use of nanoporebased metagenomic sequencing to conventional diagnostic Methods in the diagnosis of VAP. Method Patients who underwent bronchoscopy with suspicion of VAP between February 2020 and February 2021 in a medical intensive care unit of a university affiliated hospital were screened for analysis. There were 37 cases in which both the conventional diagnostic Methods and nanopore sequencing were performed using bronchial washing or bronchoalveolar lavage specimens. The nanopore sequencing Results were compared to the conventional diagnostic Methods. Results Among the 37 cases, conventional culture was positive in 12 cases and negative in 25 cases. In culture-positive cases, nanopore sequencing detected the same pathogen in 11 cases (concordance rate 91.7%). Nanopore sequencing did not detect S.maltophilia in 1 case. In culture-negative cases, nanopore sequencing also were negative in 6 cases and upper respiratory tract flora was detected in 5 cases (concordance rate 44%). Additional microbiological diagnosis was possible using nanopore in 14 cases. The additional microorganisms detected were E.faecium, A.baumanni, S,maltophilia, P.jirovecii, E.coli, S.aureus, L.pneumophilia, etc (table 1). Conclusion In patients with clinical suspicion of VAP, nanopore sequencing of bronchoscopic specimens showed high concordance rate with the conventional diagnostic Methods. In culture-negative cases, additional microbiologic diagnosis was also possible using nanopore sequencing.

엑솜 염기서열 분석 방법을 이용한 단일유전자질환의 원인 유전자 발굴

이종극(Jong-Keuk Lee) 대한의학유전학회 2010 대한의학유전학회지 Vol.7 No.2

약 7,000 여개의 단일유전자질환이 보고되어 있지만 보고된 질환의 절반도 아직 원인 유전자가 밝혀지지 못한 상황이다. 그리고 기존에 밝혀진 원인 유전자의 돌연변이형들은 대부분 단백질을 코딩하는 부위의 돌연변이에 의하여 발생하고 있어서 인간 유전체에서 단백질을 코딩하는 엑손 부위만을 선별적으로 분리하여 염기서열을 분석하는 엑솜 염기서열 분석 방법은 희귀한 유전질환의 신규 원인 유전자 발굴을 위한 매우 효과적인 유전 분석법이 될 것이다. 엑솜은 전체 유전체의 약 1.5% 정도를 차지하고 있어서 매우 경제적으로 분석이 가능하다. 그리고 엑솜 염기서열 분석 방법은 엑솜 부위를 선별하는 기술과 대용량 염기서열 분석기술로 수행된다. Freeman-Sheldon 증후군의 원인 유전자를 엑솜 염기서열 분석 방법으로 발굴한 이후로 단일유전자질환의 원인 유전자 발굴을 위한 표준 분석법으로 엑솜 염기서열 분석 방법이사용되고 있다. 향후에는 엑솜 염기서열 분석 방법이 다양한 복합질병의 유전분석에도 활용되어 개인 맞춤의학의 실현을 앞당기는데 크게 기여할 것으로 기대된다. More than 7,000 rare Mendelian diseases have been reported, but less than half of all rare monogenic disorders has been discovered. In addition, the majority of mutations that are known to cause Mendelian disorders are located in protein-coding regions. Therefore, exome sequencing is an efficient strategy to selectively sequence the coding regions of the human genome to identify novel genes associated with rare genetic disorders. The “exome” represents all of the exons in the human genome, constituting about 1.5% of the human genome. Exome sequencing is carried out by targeted capture and intense parallel sequencing. After the first report of successful exome sequencing for the identification of causal genes and mutations in Freeman Sheldon syndrome, exome sequencing has become a standard approach to identify genes in rare Mendelian disorders. Exome sequencing is also used to search the causal genes and variants in complex diseases. The successful use of exome sequencing in Mendelian disorders and complex diseases will facilitate the development of personalized genomic medicine.

Single-cell and spatial sequencing application in pathology

김윤섭,최진용,이석형 대한병리학회 2023 Journal of Pathology and Translational Medicine Vol.57 No.1

Traditionally, diagnostic pathology uses histology representing structural alterations in a disease’s cells and tissues. In many cases, however, it is supplemented by other morphology-based methods such as immunohistochemistry and fluorescent in situ hybridization. Single-cell RNA sequencing (scRNA-seq) is one of the strategies that may help tackle the heterogeneous cells in a disease, but it does not usually provide histologic information. Spatial sequencing is designed to assign cell types, subtypes, or states according to the mRNA expression on a histological section by RNA sequencing. It can provide mRNA expressions not only of diseased cells, such as cancer cells but also of stromal cells, such as immune cells, fibroblasts, and vascular cells. In this review, we studied current methods of spatial transcriptome sequencing based on their technical backgrounds, tissue preparation, and analytic procedures. With the pathology examples, useful recommendations for pathologists who are just getting started to use spatial sequencing analysis in research are provided here. In addition, leveraging spatial sequencing by integration with scRNA-seq is reviewed. With the advantages of simultaneous histologic and single-cell information, spatial sequencing may give a molecular basis for pathological diagnosis, improve our understanding of diseases,

Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of the BRAFV600E Mutation in Thyroid Neoplasm

오혜선,권혜미,박수연,김미진,전민지,김태용,송영기,김원배,최진,김원구,송동은 대한내분비학회 2018 Endocrinology and metabolism Vol.33 No.1

Background: The BRAFV600E mutation is the most common genetic alteration identified in papillary thyroid carcinoma (PTC). Becauseof its costs effectiveness and sensitivity, direct Sanger sequencing has several limitations. The aim of this study was to evaluatethe efficiency of immunohistochemistry (IHC) as an alternative method to detect the BRAFV600E mutation in preoperative and postoperativetissue samples. Methods: We evaluated 71 patients who underwent thyroid surgery with the result of direct sequencing of the BRAFV600E mutation. IHC staining of the BRAFV600E mutation was performed in 49 preoperative and 23 postoperative thyroid specimens. Results: Sixty-two patients (87.3%) had PTC, and of these, BRAFV600E was confirmed by direct sequencing in 57 patients (91.9%). In 23 postoperative tissue samples, the BRAFV600E mutation was detected in 16 samples (70%) by direct sequencing and 18 samples(78%) by IHC. In 24 fine needle aspiration (FNA) samples, BRAFV600E was detected in 18 samples (75%) by direct sequencing and16 samples (67%) by IHC. In 25 core needle biopsy (CNB) samples, the BRAFV600E mutation was detected in 15 samples (60%) bydirect sequencing and 16 samples (64%) by IHC. The sensitivity and specificity of IHC for detecting the BRAFV600E mutation were77.8% and 66.7% in FNA samples and 99.3% and 80.0% in CNB samples. Conclusion: IHC could be an alternative method to direct Sanger sequencing for BRAFV600E mutation detection both in postoperativeand preoperative samples. However, application of IHC to detect the BRAFV600E mutation in FNA samples is of limited valuecompared with direct sequencing.

Type-specific Amplification of 5S rRNA from Panax ginseng Cultivars Using Touchdown (TD) PCR and Direct Sequencing

Hun Sun,Hong-Tao Wang,Woo-Saeng Kwon,Yeon-Ju Kim,Deok-Chun Yang 고려인삼학회 2009 Journal of Ginseng Research Vol.33 No.1

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.