Genomic analysis in genes responsible for eye abnormalities in patients

导致患者眼睛异常的基因的基因组分析

• 批准号: 9362394
• 负责人: Xinjing Wang
• 金额: $ 52.48万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9362394
• 关键词: Algorithms; Alleles; American; Amino Acids; Bioinformatics; Biological Assay; Biological Sciences; CLIA certified; Categories; Chromosome abnormality; Clinic; Clinical; Clinical Trials; Code; Communities; Complementary DNA; Computer software; Corneal dystrophy; Custom; DNA; DNA Resequencing; Data; Deletion Mutation; Detection; Development; Diagnostic; Disease; Enrollment; Epigenetic Process; Eye; Eye Abnormalities; Eye diseases; FDA approved; Family; Frequencies; Gene Deletion; Genes; Genome; Genomic Segment; Genomics; Genotype; Goals; Guidelines; Hereditary Disease; High-Throughput Nucleotide Sequencing; Hour; Housing; Human Genome; Inherited; Laboratories; Lead; Manuscripts; Medical Genetics; Melissa; Methodology; Methods; Modification; Molecular; Molecular Diagnostic Testing; Mutation; Mutation Analysis; Nucleotides; Oculocutaneous Albinism; Participant; Pathogenicity; Patients; Peer Review; Phenotype; Population; Preparation; Procedures; Process; Protocols documentation; Publications; Publishing; RNA; RNA Splicing; RNA library; Reading; Recording of previous events; Regulation; Reporting; Research; Retinal; Retinal Dystrophy; Running; Sampling; Sequence Analysis; Serum; Services; Site Visit; Specific qualifier value; Staging; Technology; Testing; Time; Validation; Variant; Work; X-Linked Retinoschisis; Yang; Zein; adeno-associated viral vector; base; cDNA Library; college; computerized data processing; cost; cost effective; deep sequencing; design; disease-causing mutation; exome; exome sequencing; genome sequencing; genomic variation; improved; indexing; interest; macular dystrophy; molecular diagnostics; next generation sequencing; novel strategies; operation; programs; protein structure; research and development; screening; segregation; sequencing platform; targeted sequencing; tool; Algorithms; Alleles; American; Amino Acids; Bioinformatics; Biological Assay; Biological Sciences; CLIA certified; Categories; Chromosome abnormality; Clinic; Clinical; Clinical Trials; Code; Communities; Complementary DNA; Computer software; Corneal dystrophy; Custom; DNA; DNA Resequencing; Data; Deletion Mutation; Detection; Development; Diagnostic; Disease; Enrollment; Epigenetic Process; Eye; Eye Abnormalities; Eye diseases; FDA approved; Family; Frequencies; Gene Deletion; Genes; Genome; Genomic Segment; Genomics; Genotype; Goals; Guidelines; Hereditary Disease; High-Throughput Nucleotide Sequencing; Hour; Housing; Human Genome; Inherited; Laboratories; Lead; Manuscripts; Medical Genetics; Melissa; Methodology; Methods; Modification; Molecular; Molecular Diagnostic Testing; Mutation; Mutation Analysis; Nucleotides; Oculocutaneous Albinism; Participant; Pathogenicity; Patients; Peer Review; Phenotype; Population; Preparation; Procedures; Process; Protocols documentation; Publications; Publishing; RNA; RNA Splicing; RNA library; Reading; Recording of previous events; Regulation; Reporting; Research; Retinal; Retinal Dystrophy; Running; Sampling; Sequence Analysis; Serum; Services; Site Visit; Specific qualifier value; Staging; Technology; Testing; Time; Validation; Variant; Work; X-Linked Retinoschisis; Yang; Zein; adeno-associated viral vector; base; cDNA Library; college; computerized data processing; cost; cost effective; deep sequencing; design; disease-causing mutation; exome; exome sequencing; genome sequencing; genomic variation; improved; indexing; interest; macular dystrophy; molecular diagnostics; next generation sequencing; novel strategies; operation; programs; protein structure; research and development; screening; segregation; sequencing platform; targeted sequencing; tool

To evaluate the newly developed next generation sequencing assay by analyzing patients with isolated eye abnormalities collected in earlier studies including patients enrolled through eyeGENE program: This project began several years ago in the OGFVB/NEI. We first developed a screening tool by the resequencing CHIP on 93 genes. Later, we developed another approach using microdroplet PCR technology and the next generation sequencing (NGS) to analyze 184 genes. Our manuscript of the microdroplet PCR technology has been submitted. Later on, we adapted a target capture plus NGS technology. We developed a new panel composed of 330 genes related to retinal function and development. Using NGS technology, we have been more actively analyzing patient with variety categories of retinal dystrophies in a more efficient and accurate approach. We have performed NGS sequencing for more than 350 samples with a variety of ophthalmic diseases. We are continuing the procedures to analyze more patients with RP. Meanwhile, we have built our own in house bioinformatics pipeline to analyze NGS data. We are capable to analyze all kinds of NGS data, including WES data. Using this pipeline, we have built our own bioinformatics pipeline and have analyzed NGS data from different projects. To date, we have analyzed over 300 samples for several projects including the following: o Perform analysis for NISC project. Assisted in the development of the pipeline of 100 samples to be analyzed from NISC. o Perform analysis for whole exome sequencing project (WES). Analyzed samples and developed protocol for whole exome sequencing analysis. We also used these samples to search for ACMG common genes. We have also purchased a software package for mutation analysis, Alamut. This software assists us in the determination of variant pathogenicity. Alamut consolidates data from 3 mutation algorithms, population frequencies, nucleotide and amino acid conservation, protein structures, and splicing information into one platform. This saves the DDL lab significant time and effort when analyzing variants. We created a custom DDL Variant Report to use with Alamut for variant analysis. The report provides easy access to variant information without the need for special software. o Improved overall sequencing quality. Initially, we loaded our sequencing runs at 20pM. As of now, we are loading all of our sequencing runs at 10pM. Doing so increases the number of reads per sample while improving the amount of coverage that is received for each sample. The overall quality of the runs has improved as well; specifically, Q30 scores and Cluster Pass Filtering Scores. o Continuously working to develop all Standard Operating Procedures in accordance with CLIA regulations, so the Next-Generation sequencing protocol can become clinical procedure. Other projects: To develope a RNA/cDNA Sure Select Targeted Enrichment project. Starting a project with Agilent for Strand-specific RNA library preparation and target enrichment. o Determining the number of samples and parameters needed for optimal clustering o Adjusting the loading concentration of the sequencing run based on our sequencing needs o Pooling the samples together at specified concentration(10nM) o Manually correcting index sequences o Adjusting the sequencing parameters: Read1:151 cycles, Read2:151, Index:8bp o Perform analysis and data processing Through our successful sequencing, we are now attempting to sequence the entire cDNA library using the same parameters: Although, additional testing and troubleshooting will be needed in order to complete this project. Proposed a new method to develop a new sequencing platform covering all genes known to cause inherited eye diseases by deep sequencing. We are currently testing a new method provided by Agilent Technologies that will allow us to provide a superior method for comprehensive and efficient targeted sequencing that offers cost efficiencies and a streamlined workflow; while, accelerating the turn-around time from sample to date and providing deep coverage of genomic regions of interest. This project will allow us to potentially decrease the turnaround time frame <2 weeks /16 samples along with the cost per sample to $284. So far, we have processed a total of 36 samples using this method. New approach to perform NGS sequencing We are currently testing a new method provided by Agilent Technologies that will allow us to provide a superior method for comprehensive and efficient targeted sequencing that offers cost efficiencies and a streamlined workflow; while, accelerating the turn-around time from sample to date and providing deep coverage of genomic regions of interest. This project will allow us to potentially decrease the turnaround time frame <2 weeks /16 samples along with the cost per sample to $284. Clinical laboratory for FDA approved clinical trial, XLRS2 project We are the clinical laboratory for the XLRS2 clinical trial by processed 77 Serum samples, 9 AAV Vector Dilution Storage, 3 FDA site visits, and 1 shipment for clinical analysis to the Advanced Bioscience Laboratories, Inc (ABL) in support of XLRS2 clinical trial. It included performing 77 serum extractions on nine participants and stored 9 AAV vector dilutions. Time spent processing each serum sample is approximately 45 minutes to 1 hour. Preparing publications for current collaborative research DDL commits to publish our research and our collaborative research results. Here is the list of our currently manuscripts in different stages: 1. Xiaodong Jiao Anren Li, Zi-Bing Jin, Xinjing Wang, Alessandro Iannaccone, Elias Traboulsi, Michael B. Gorin, Francesca Simonelli, and J. Fielding Hejtmancik* Identification and Population History of CYP4V2 mutations in Patients with Bietti Crystalline Corneoretinal Dystrophy (post peer-review resubmission) 2. Annalisa Nicoletti, Lucia Ziccardi, Paolo Enrico Maltese, Sabrina Benedetti, Orazio Palumbo, Michelina Rendina, Leonardo DAgruma, Benedetto Falsini, Xinjing Wang, Matteo Bertelli*. Design and validation of a new MLPA-based assay for the detection of RS1 gene deletions and application in a large family with X-linked juvenile Retinoschisis (post peer-review revision) 3. Jackson Gao, Leera DSounza, Keith Wetherby, Christian Antolik, Melissa Reeves, David Adams, Xinjing Wang* Retrospective Mutation Analysis in Oculocutaneous Albinism Patients for a 2.7 kb deletion mutation in the OCA2 gene revealed a co-segregation of the controversial allele, p.R305W (post peer-review revision). 4. Xinjing Wang*, Wadih Zein, Leera DSouza, Chimere Roberson, Keith Wetherby, Hong He, Angela Villarta, Delphine Blaine, Amy Turriff, Kory Johnson, Yang C Fann. Applying the next generation sequencing by microdroplet PCR to determine the disease causing mutations in retinal dystrophies (submitted). 5. Xinjing Wang*, Danyao Nie, Chimere Roberson, Keith Wetherby, Angela Villarta, Kerry Goetz, Alexandra Garafalo, Rebecca Parrish, Santa Tumminia, Kory Johnson, Yang C Fann. NGS Analysis of Corneal Dystrophy Related Genes Using Modified Microdroplet PCR Protocol in Patients with Corneal Dystrophies (manuscript in preparation). 6. Xinjing Wang*, Chimere Roberson, Keith Wetherby, Melissa Reeves, Wadih Zein, Delphine Blain, Amy Turriff, Robert Hufnagel, Santa J. Tumminia, Brian Brook. NGS analysis of 100 samples from the eyeGENE program to determine the disease causing mutations in retinal dystrophies (manuscript in preparation).

为了评估新开发的下一代测序测定法，通过分析早期研究中收集的孤立眼睛异常的患者，包括通过Eyegene计划入学的患者： 该项目始于几年前的OGFVB/NEI。我们首先通过对93个基因的重新配置芯片开发了一种筛选工具。后来，我们开发了另一种使用微副PCR技术和下一代测序（NGS）来分析184个基因的方法。 我们已经提交了我们的微螺旋PCR技术手稿。 后来，我们改编了目标捕获和NGS技术。 我们开发了一个由330个与视网膜功能和发育相关的基因组成的新面板。使用NGS技术，我们正在以更高效，更准确的方法进行更积极地分析各种视网膜营养不良的患者。 我们已经为350多种眼科疾病进行了350多个样本进行了NGS测序。 我们正在继续分析更多RP患者的程序。 同时，我们已经建立了自己的内部生物信息学管道来分析NGS数据。我们能够分析包括WES数据在内的各种NGS数据。使用此管道，我们建立了自己的生物信息学管道，并分析了来自不同项目的NGS数据。迄今为止，我们已经分析了300多个样本针对多个项目，包括以下几个项目： o对NISC项目执行分析。 协助开发了从NISC分析的100个样品的管道。 o对整个外显子测序项目（WES）执行分析。 分析了样品并开发了用于整个外显子组测序分析的方案。我们还使用这些样品搜索ACMG共同基因。 我们还购买了用于突变分析的软件包，Alamut。该软件有助于我们确定变异的致病性。 Alamut从3种突变算法，种群频率，核苷酸和氨基酸保护，蛋白质结构以及将信息拼接到一个平台中的数据巩固了数据。在分析变体时，这节省了DDL实验室的大量时间和精力。 我们创建了一个自定义DDL变体报告，用于与Alamut一起用于变体分析。该报告无需特殊软件即可轻松访问变体信息。 o提高了整体测序质量。 最初，我们在晚上20点加载了测序运行。截至目前，我们正在加载所有测序在晚上10点运行。这样做会增加每个样本的读数数量，同时改善每个样本收到的覆盖范围。 跑步的总体质量也有所提高。具体而言，Q30分数和群集通过过滤得分。 o不断努力根据CLIA法规制定所有标准的操作程序，因此下一代测序方案可以成为临床程序。 其他项目： 为了开发RNA/cDNA肯定选择的靶向富集项目。 使用安捷伦（Agilent）开始一个项目，以进行链特异性RNA库制备和目标富集。 o确定最佳聚类所需的样本数量和参数 o根据我们的测序需求调整测序运行的负载浓度 o以指定浓度（10nm）将样品汇总在一起 o手动纠正索引序列 o调整测序参数：read1：151循环，read2：151，索引：8bp o执行分析和数据处理 通过我们的成功测序，我们现在正在尝试使用相同的参数对整个cDNA库进行测序：尽管需要进行其他测试和故障排除以完成此项目。 提出了一种新的方法来开发一个新的测序平台，该平台涵盖了所有已知通过深度测序引起遗传性眼部疾病的基因。我们目前正在测试Agilent Technologies提供的一种新方法，该方法将使我们能够为综合有效的目标测序提供卓越的方法，以提供成本效率和简化的工作流程；同时，加速了从样本到日期的周转时间，并提供了对基因组感兴趣的基因组区域的深入覆盖。该项目将使我们能够减少周转时间范围<2周 /16个样本，每样本成本至284美元。 到目前为止，我们已经使用此方法处理了总共36个样本。 执行NGS测序的新方法 我们目前正在测试Agilent Technologies提供的一种新方法，该方法将使我们能够为综合有效的目标测序提供卓越的方法，以提供成本效率和简化的工作流程；同时，加速了从样本到日期的周转时间，并提供了对基因组感兴趣的基因组区域的深入覆盖。该项目将使我们能够减少周转时间范围<2周 /16个样本，每样本成本至284美元。 FDA批准的临床实验室XLRS2项目 我们是通过加工的77种血清样品，9个AAV载体稀释储存，3次FDA现场访问和1次运送临床分析的临床试验的临床实验室，用于支持XLRS2临床试验。 它包括对9名参与者进行77种血清提取物，并储存9个AAV载体稀释液。处理每个血清样品花费的时间约为45分钟至1小时。 为当前的合作研究准备出版物 DDL致力于发布我们的研究和我们的协作研究结果。 这是我们目前在不同阶段的手稿的列表： 1. Xiaodong Jiao Anren Li, Zi-Bing Jin, Xinjing Wang, Alessandro Iannaccone, Elias Traboulsi, Michael B. Gorin, Francesca Simonelli, and J. Fielding Hejtmancik* Identification and Population History of CYP4V2 mutations in Patients with Bietti Crystalline Corneoretinal Dystrophy (post peer-review resubmission) 2。AnnalisaNicoletti，Lucia Ziccardi，Paolo Enrico Maltese，Sabrina Benedetti，Orazio Palumbo，Michelina Rendina，Leonardo Dagruma，Benedetto Falsini，Xinjing Wang，Matteo Bertelli*。 设计和验证基于MLPA的新测定法，用于检测RS1基因删除和在一个具有X连锁少年视网膜的大家庭中（PEER-PEER-REVIEW REVISION） 3. Jackson Gao, Leera DSounza, Keith Wetherby, Christian Antolik, Melissa Reeves, David Adams, Xinjing Wang* Retrospective Mutation Analysis in Oculocutaneous Albinism Patients for a 2.7 kb deletion mutation in the OCA2 gene revealed a co-segregation of the controversial allele, p.R305W (post peer-review revision). 4。XinjingWang*，Wadih Zein，Leera Dsouza，Chimere Roberson，Keith Wetherby，Hong He，Angela Villarta，Delphine Blaine，Amy Turriff，Kory Johnson，Yang C Fann。通过微圆珠PCR应用下一代测序，以确定引起视网膜营养不良突变的疾病（已提交）。 5。XinjingWang*，Danyao Nie，Chimere Roberson，Keith Wetherby，Angela Villarta，Kerry Goetz，Alexandra Garafalo，Rebecca Parrish，Santa Tumminia，Kory Johnson，Yang C Fann。使用修饰的微核PCR方案对角膜营养不良的基因进行NGS分析（制剂中的手稿）。 6。XinjingWang*，Chimere Roberson，Keith Wetherby，Melissa Reeves，Wadih Zein，Delphine Blain，Amy Turriff，Robert Hufnagel，Santa J. Tumminia，Brian Brook。 NGS对眼睛金油管计​​划的100个样本的分析，以确定引起视网膜营养不良突变的疾病（制备中的手稿）。