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Confidence intervals for Markov chain transition probabilities based on Next generation sequencing reads data

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Quantitative Biology 2020年第2期8卷 143-154页

作者： Lin Wan Xin Kang Jie Ren Fengzhu Sun NCMIS LSCAcademy of Mathematics and Systems ScienceChinese Academy of SciencesBeijing 100190China School of Mathematical Sciences Fudan UniversityShanghai 200433China Quantitative and Computational Biology Program University of Southern CaliforniaLos AngelesCA 90089USA

Background:Markov chains(MC)have been widely used to model molecular sequences.The estimations of MC transition matrix and confidence intervals of the transition probabilities from long sequence data have been intensively studied in the past decades.In Next generation sequencing(NGS),a large amount of short reads are generated.These short reads can overlap and some regions of the genome may not be sequenced resulting in a new type of data.Based on NGS data,the transition probabilities of MC can be estimated by moment estimators.However,the classical asymptotic distribution theory for MC transition probability estimators based on long sequences is no longer valid.Methods:In this study,we present the asymptotic distributions of several statistics related to MC based on NGS data.We show that,after scaling by the effective coverage d defined in a previous study by the authors,these statistics based on NGS data approximate to the same distributions as the corresponding statistics for long sequences.Results:We apply the asymptotic properties of these statistics for finding the theoretical confidence regions for MC transition probabilities based on NGS short reads data.We validate our theoretical confidence intervals using both simulated data and real data sets,and compare the results with those by the parametric bootstrap method.Conclusions:We find that the asymptotic distributions of these statistics and the theoretical confidence intervals of transition probabilities based on NGS data given in this study are highly accurate,providing a powerful tool for NGS data analysis.

关键词： Markov chains Next generation sequencing transition probabilities confidence intervals

Application of Next generation sequencing technology on contamination monitoring in microbiology laboratory

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Biosafety and Health 2019年第1期1卷 25-31页

作者： Yan Xiao Li Zhang Bin Yang Mingkun Li Lili Ren Jianwei Wang NHC Key Laboratory of Systems Biology of Pathogens Institute of Pathogen BiologyChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100730China Key Laboratory of Genomic and Precision Medicine Beijing Institute of GenomicsChinese Academy of SciencesBeijing 100101China Center for Excellence in Animal Evolution and Genetics Chinese Academy of SciencesKunming 650223China

The surveillance and prevention of pathogenic microbiological contamination are the most important tasks of biosafety management in the lab.There is an urgent need to establish an effective and unbiased method to evaluate and monitor such contamination.This study aims to investigate the utility of Next generation sequencing(NGS)method to detect possible contamination in the microbiology laboratory.Environmental samples were taken at multiple sites at the lab including the inner site of centrifuge rotor,the bench used for molecular biological tests,the benches of biosafety cabinets used for viral culture,clinical sample pre-treatment and nucleic acids extraction,by scrubbing the sites using sterile flocked swabs.The extracted total nucleic acids were used to construct the libraries for deep sequencing according to the protocol of Ion Torrent platform.At least 1G raw data was obtained for each sample.The reads of viruses and bacteria accounted for 0.01±0.02%,and 77.76±12.53%of total reads respectively.The viral sequences were likely to be derived from gene amplification products,the nucleic acids contaminated in fetal bovine serum.Reads from environmental microorganisms were also identified.Our results suggested that NGS method was capable of monitoring the nucleic acids contaminations from different sources in the lab,demonstrating its promising utility in monitoring and assessing the risk of potential laboratory contamination.The risk of contamination from reagents,remnant DNA and environment should be considered in data analysis and results interpretation.

关键词： Laboratory contamination Biosafety Next generation sequencing Deep sequencing

Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing:A cohort analysis

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World Journal of Gastroenterology 2023年第18期29卷 2864-2874页

作者： Stefania Bunduc Bianca Varzaru Razvan Andrei Iacob Andrei Sorop Ioana Manea Andreea Spiridon Raluca Chelaru Adina Emilia Croitoru Gabriel Becheanu Mona Dumbrava Simona Dima Irinel Popescu Cristian Gheorghe Faculty of Medicine Carol Davila University of Medicine and PharmacyBucharest 020021Romania Digestive Diseases and Liver Transplantation Center Fundeni Clinical InstituteBucharest 022328Romania Center of Excellence in Translational Medicine Fundeni Clinical InstituteBucharest 022328Romania

BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer.For genotyping aimed samples current guidelines recommend using core specimens,although based on moderate quality evidence.However,in clinical practice among the endoscopic ultrasound(EUS) guided tissue acquisition methods,fine needle aspiration(FNA) is the most widely performed.AIM To assess the adequacy for Next generation sequencing(NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma(PDAC) samples.METHODS Between November 2018 and December 2021,105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center.Either 22 gauge(G) or 19G FNA needles were used.One pass was dedicated to DNA extraction.DNA concentration and purity(A260/280,A260/230) were assessed by spectrophotometry.We assessed the differences in DNA parameters according to needle size and tumor characteristics(size,location) and the adequacy of the extracted DNA for NGS(defined as A260/280 ≥ 1.7,and DNA yield:≥ 10 ng for amplicon based NGS,≥ 50 ng for whole exome sequencing [WES],≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and ttest respectively.Moreover,we compared DNA purity parameters across the different DNA yield categories.RESULTS Our cohort included 49% male patients,aged 67.02 ± 8.38 years.The 22G needle was used in 71%of the cases.The DNA parameters across our samples varied as follows:DNA yield:1289 ng(inter quartile range:534.75-3101),A260/280 = 1.85(1.79-1.86),A260/230 = 2.2(1.72-2.36).DNA yield was > 10 ng in all samples and > 100 ng in 93% of them(one sample 100

关键词： Pancreatic adenocarcinoma Endoscopic ultrasound guided fine needle aspiration Next generation sequencing DNA yield Needle size Genetic testing

A novel PIK3CD C896T mutation detected in bilateral sudden sensorineural hearing loss using Next generation sequencing:An indication of primary immunodeficiency

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Journal of Otology 2016年第2期11卷 78-83页

作者： Jing Zou Xiangqiang Duan Guiliang Zheng Zhen Zhao Shiyue Chen Pu Dai Hongliang Zheng Department of Otolaryngology-Head and Neck Surgery Center for Otolaryngology-Head & Neck Surgery of Chinese PLA Changhai Hospital Second Military Medical University Department of Radiology National Key Discipline Changhai Hospital Second Military Medical University Department of Otolaryngology-Head and Neck Surgery Chinese PLA General Hospital

Objective:To investigate immune-related genetic background in bilateral sudden sensorineural hearing loss (SSNHL). Case report and methods: The case is a 45-year-old man presenting with a 7-year history of bilateral profound SSNHL. Blood biochemical testing demonstrated increased levels of total cholesterol (5.88 mmol/L). Tests for hepatitis B showed a positive antibody against the hepatitis B core antigen. Complement C3 was below the normal value, and complement C4 and IgG were in the lower range of normal values. CT images showed a normal inner ear and vestibular aqueduct but round window membranous ossification on both sides. A total number of 232 immune-associated genes were sequenced using the Next generation sequencing technique. Results: Mutations were detected in 5 genes, including the phosphoinositide 3-kinase catalytic subunit delta (PIK3CD), caspase recruitment domain-containing protein 9 (CARD9), complement factor H-related (CFHR2), immunoglobulin lambda-like polypeptide 1 Protein (IGLL1), and transmembrane channel-like gene family 8 (TMC8). In the PIK3CD gene, a C896T substitute in exon 7 was detected. This mutation causes primary immunodeficiency and is an autosomal dominant disease. Conclusion: The PIK3CD C896T mutation responsible for primary immunodeficiency may contribute to the onset of bilateral SSNHL with subsequent rapid progression.

关键词： Sudden sensorineural hearing loss Immunology Genetics Next generation sequencing

Fecal gene detection based on Next generation sequencing for colorectal cancer diagnosis

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World Journal of Gastroenterology 2022年第25期28卷 2920-2936页

作者： Si-Yu He Ying-Chun Li Yong Wang Hai-Lin Peng Cheng-Lin Zhou Chuan-Meng Zhang Sheng-Lan Chen Jian-Feng Yin Mei Lin Department of Clinical Laboratory Taizhou People's Hospital(Postgraduate Training Base of Dalian Medical University)Taizhou 225300Jiangsu ProvinceChina Department of Clinical Laboratory The First People's Hospital of Tianmen CityTianmen 431700Hubei ProvinceChina Department of General Surgery Taizhou People's Hospital(Postgraduate Training Base of Dalian Medical University)Taizhou 225300Jiangsu ProvinceChina Central Laboratory Taizhou People's Hospital(Postgraduate training base of Dalian Medical University)Taizhou 225300Jiangsu ProvinceChina Department of Laboratory Taizhou Genewill Medical Laboratory Company LimitedTaizhou 225300Jiangsu ProvinceChina Department of Laboratory Jiangsu CoWin Biotech Co.Ltd.Taizhou 225300Jiangsu ProvinceChina

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.Given its insidious onset,the condition often already progresses to advanced stage when symptoms occur.Thus,early diagnosis is of great significance for timely clinical intervention,efficacy enhancement,and prognostic improvement.Featuring high throughput,fastness,and rich information,Next generation sequencing(NGS)can greatly shorten the detection time,which is a widely used detection technique at present.AIM To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC,and to establish a technological platform for CRC screening,diagnosis,and efficacy monitoring through fecal DNA detection.METHODS NGS was used to sequence the stool DNA of patients with CRC,which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease,as well as the tumor tissues of CRC patients.Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened,and their significances in diagnosing CRC and predicting patients'prognosis were comprehensively evaluated.RESULTS High mutation frequencies of TP53,APC,and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery.Contrastively,no pathogenic mutations of the above three genes were noted in the postoperative stools,the normal controls,or the benign intestinal disease group.This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients.The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent.Compared to the postoperative stools and the stools in the two control groups,the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools(χ^(2)=7.328,P<0.05;χ^(2)=4.219,P<0.05),suggesting that fecal TP53 and KRAS genes can be used for CRC screening,diagnosis,and

关键词： Colorectal cancer Feces Next generation sequencing Diagnosis Gene

De novo assembly of transcriptome from next-generation sequencing data

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Frontiers of Electrical and Electronic Engineering in China 2016年第2期4卷 94-105页

作者： Xuan Li Yimeng Kong Qiong-Yi Zhao Yuan-Yuan Li Pei Hao Key Laboratory of Synthetic Biology CAS Center for Excellence in Molecular Plant Sciences Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China The Ugniversitvof Queensland. Queensland Brain Institute. St Lucia. QI'D 4072. Ausiralia Shanghai Center for Bioinformation Technology Shanghai Industrial Technology Institute Shanghai 201203 China Key Laboratory of Molecular Virology and Immunology institute Pasteur of Shanghai Chinese Academy of SciencesShanghai 200031 China

Reconstruction of transcriptome by de novo assembly from Next generation sequencing （NGS） short-sequence reads provides an essential mean to catalog expressed genes, identify splicing isoforms, and capture the expression detail of transcripts for organisms with no reference genome available. De novo transcriptome assembly faces many unique challenges, including alternative splicing, variable expression level covering a dynamic range of several orders of magnitude, artifacts introduced by reverse transcription, etc. In the current review, we illustrate the grand strategy in applying De Bruijn Graph （DBG） approach in de novo transcriptome assembly. We further analyze many parameters proven critical in transcriptome assembly using DBG. Among them, k-met length, coverage depth of reads, genome complexity, performance of different programs are addressed in greater details. A multi-k-mer strategy balancing efficiency and sensitivity is discussed and highly recommended for de novo transcriptome assembly. Future direction points to the combination of NGS and third generation sequencing technology that would greatly enhance the power of de novo transcriptomics study.

关键词： transcriptome de novo assembly De Bruijn Graph Next generation sequencing k-mer length RNA splicing performance

Comparison of the experimental methods in haplotype sequencing via Next generation sequencing

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Frontiers of Electrical and Electronic Engineering in China 2016年第2期4卷 106-114页

作者： Jing Tu Na Lu Mengqin Duan An Ju Xiao Sun Zuhong Lu State Key Laboratory of Bioelectronics Southeast University Nanjing 210096 China

Although the diploid nature has been observed for over 50 years, phasing the diploid is still a laborious task. The speed and throughput of Next generation sequencing have largely increased in the past decades. However, the short read-length remains one of the biggest challenges of haplotype analysis. For instance, reads as short as 150 bp span no more than one variant in most cases. Numerous experimental technologies have been developed to overcome this challenge. Distance, complexity and accuracy of the linkages obtained are the main factors to evaluate the efficiency of whole genome haplotyping methods. Here, we review these experimental technologies, evaluating their efficiency in linkages obtaining and system complexity. The technologies are organized into four categories based on its strategy：（i） chromosomes separation, （ii） dilution pools, （iii） crosslinking and proximity ligation, （ix） long-read technologies. Within each category, several subsections are listed to classify each technology. Innovative experimental strategies are expected to have high-quality performance, low cost and be labor-saving, which will be largely desired in the future.

关键词： Next generation sequencing haplotyping haplotype sequencing

next generation Transcriptome sequencing and Quantitative Real-Time PCR Technologies for Characterisation of the Bemisia tabaci Asia 1 mtCOI Phylogenetic Clade

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Journal of Integrative Agriculture 2012年第2期11卷 281-292页

作者： Susan Seal Mitulkumar V Patel Carl Collins John Colvin David Bailey Natural Resources Institute University of GreenwichChatham Maritime Kent ME4 4TBUnited Kingdom

A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.

关键词： Bemisia tabaci whitefly transcriptome Next generation sequencing quantitative real-time （QRT）-PCR Asia 1 mtCOI

The role of gene variants in the pathogenesis of neurodegenerative disorders as revealed by Next generation sequencing studies:a review

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Translational Neurodegeneration 2017年第1期6卷 245-255页

作者： Shirley Yin-Yu Pang Kay-Cheong Teo Jacob Shujui Hsu Richard Shek-Kwan Chang Miaoxin Li Pak-Chung Sham Shu-Leong Ho Division of Neurology Department of MedicineQueen Mary HospitalUniversity of Hong KongHong KongPeople’s Republic of China Centre for Genomic Sciences University of Hong KongHong KongPeople’s Republic of China Department of Medical Genetics Zhongshan School of MedicineSun Yat-sen UniversityGuangzhouPeople’s Republic of China Key Laboratory of Tropical Disease Control(SYSU) Ministry of EducationGuangzhouPeople’s Republic of China

The clinical diagnosis of neurodegenerative disorders based on phenotype is difficult in heterogeneous conditions with overlapping symptoms.It does not take into account the disease etiology or the highly variable clinical course even amongst patients diagnosed with the same disorder.The advent of Next generation sequencing(NGS)has allowed for a system-wide,unbiased approach to identify all gene variants in the genome simultaneously.With the plethora of new genes being identified,genetic rather than phenotype-based classification of Mendelian diseases such as spinocerebellar ataxia(SCA),hereditary spastic paraplegia(HSP)and Charcot-Marie-Tooth disease(CMT)has become widely accepted.It has also become clear that gene variants play a role in common and predominantly sporadic neurodegenerative diseases such as Parkinson’s disease(PD)and amyotrophic lateral sclerosis(ALS).The observation of pleiotropy has emerged,with mutations in the same gene giving rise to diverse phenotypes,which further increases the complexity of phenotype-genotype correlation.Possible mechanisms of pleiotropy include different downstream effects of different mutations in the same gene,presence of modifier genes,and oligogenic inheritance.Future directions include development of bioinformatics tools and establishment of more extensive public genotype/phenotype databases to better distinguish deleterious gene variants from benign polymorphisms,translation of genetic findings into pathogenic mechanisms through in-vitro and in-vivo studies,and ultimately finding disease-modifying therapies for neurodegenerative disorders.

关键词： Next generation sequencing Neurodegenerative diseases Genetics Pleiotropy