遗传病二代测序临床检测全流程规范化共识探讨(3)——数据分析流程

基因组测序数据的生物信息学分析以及变异的临床相关性解读是基于二代测序的遗传病诊断全流程的核心内容。本文探讨了生物信息学分析、数据存储和数据解读环节的流程、功能、主要参数指标及建议等,经行业内临床医师、检测实验室等相关专家讨论,对符合孟德尔遗传规律的胚系突变的遗传病规范化检测达成了共识,以期为提髙基于二代测序的遗传病诊断的同质性和可靠性提供指导。     

遗传病二代测序临床检测全流程规范化共识探讨(1)——遗传检测前流程

遗传检测前的准备工作是基因检测的基础和出发点,其流程包括临床信息收集、检测方案拟定、检测前遗传咨询以及知情同意书和检测委托书的填写等。临床若能有效识别出遗传性疾病,可极大提高二代测序的诊断率,从而降低医疗成本,提高临床诊疗的功效。二代测序结果的分析在很大程度上依赖于对基因型-表型相关性的了解,全面准确地采集和评估临床表型,并用统一的标准术语来描述和记录尤为重要。不同类型的遗传病或突变种类需要用特定的检测手段方能事半功倍。检测前遗传咨询能够帮助患者及其亲属了解相关基因检测的意义并协助制定个体化的检测策略,为后续的跟踪随访奠定基础。     

遗传病二代测序临床检测全流程规范化共识探讨(4)——检测报告解读和遗传咨询

临床基因检测的结果在遗传分析环节由数据解读人员整理成一份规范的基因检测报告,提供给临床医师及受检者(若受检者有智力障碍或者为未成年人,则检测报告提供给其父母或法定监护人)。检测报告的内容应遵循相关标准及/行业共识。临床医师将综合基因检测报告及临床指征进一步诊断其所患疾病。遗传咨询师应协助临床医师及受检者或其亲属.提供检测后的遗传咨询服务。在知情同意的前提下建立检测后的跟踪随访机制。数据应由临床医疗机构与第三方检测机构共享,补充的回访数据可以帮助进一步评估报告结果,因此检测机构应定期对既往的检测报告数据及后续的回访数据进行整理分析,并同新的分析结果同步提交给患者和医疗机构。所有涉及报告、遗传咨询、后续跟踪回访及分析结果更新的活动均应遵循相关的法律法规。     

同一批次外周血BRCA基因二代测序检测出现多个相同突变的原因

<正>二代测序检测具有对一份标本同时检测多基因、多位点、多种变异类型的明显优势^[1],BRCA基因的致病性变异与乳腺癌、卵巢癌等肿瘤的发生密切相关,同时还是PARA抑制剂治疗相关的生物学标志物^[2]。针对BRCA1/2基因有无热点变异,国内对BRCA1/2基因的检测一般采用二代测序高通量测序方法^[3]。我科在日常检测工作中出现新的BRCA基因二代测序数据分析问题,现介绍如下。     

临床基因检测报告规范与基因检测行业共识探讨CSCD

二代测序技术（next generation sequencing,NGS）在临床的应用为广泛开展遗传病的基因检测、病因诊断、治疗及预防奠定了基础,但同时也伴随着一系列的问题,其中很重要的一个环节就是基因检测报告缺乏统一或基本的标准。首届“临床基因检测标准与规范专题研讨会”于2017年10月28日在深圳召开,来自全国138家机构的250多位遗传学专家、临床专家及第三方检测机构的代表共同探讨了遗传病基因检测报告的标准和规范问题。本文根据此次研讨会专家的意见和建议,就遗传病基因检测报告的原则、规范以及基因检测行业的发展进行了讨论,并发布了临床基因检测报告规范共识,以促进检测报告的规范化和标准化,推进我国基因检测行业的健康有序地发展。     

美国二代测序技术临床应用的共识声明、实践资源、技术标准和指南的概述

二代测序(next generation sequencing,NGS)的临床应用主要包括检测基因变异的全外显子组测序(whole exome sequencing,WES)和胎儿染色体非整倍体的无创产前筛查(non-invasive prenatal screening,NIPS)。本文回顾和概述了美国医学遗传与基因...     

二代测序技术检测650例非小细胞肺癌驱动基因突变

目的 探讨驱动基因在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的突变情况.方法 采用二代测序(next-generation sequencing,NGS)靶向检测,分析650例NSCLC组织中ALK、BCL2L11、BRAF、EGFR、ERBB2、FGFR、KRAS、MAP2K...     

原发性玻璃体视网膜淋巴瘤3例流式细胞术和二代测序检测分析

目的 分析原发性玻璃体视网膜淋巴瘤(primary vitreoretinal lymphoma, PVRL)的免疫和基因表型,探讨流式细胞和二代测序在PVRL诊断中的作用。方法 回顾性分析3例PVRL的细胞病理学、流式细胞、靶向二代测序或数字PCR等检测结果。结果 3例患者症状不典型,例1和例3从初诊到确诊历经1年左右。3例的流式细胞免疫分型均支持弥漫大B细胞淋巴瘤。例1细胞病理学见异常大淋巴细胞,二代测序发现MYD88 L265P和CD79B Y196H等8个基因突变。例2细胞病理学见异常幼稚大细胞,二代测序发现MYD88 L265P和CD79B Y196N等13个基因突变,房水细胞因子IL-10/IL-6升高。例3数字PCR发现房水MYD88 L265P突变。结论 流式细胞免疫分型是PVRL诊断的实用辅助手段,二代测序有利于探索新的诊断和预后标志物。     

基于二代测序的植入前遗传学检测在71例染色体易位家系中的应用

目的探讨基于二代测序(next generation sequencing,NGS)的植入前遗传学检测(preimplantation genetic test,PGT)对于染色体易位携带者实现正常生育的意义。方法对71对染色体易位携带夫妇(60例为平衡易位,11例为罗氏易位)行PGT周期的超促排卵和卵母细胞浆内单精子注射,培养至囊胚阶段,活检囊胚滋养外胚层细胞行全基因组扩增(whole genome amplification,WGA),用NGS技术对扩增产物进行基因组序列分析,选择正常或平衡胎胚植入,并对检测结果和胚胎着床、妊娠情况进行分析。结果71对夫妇共进行92个周期,活检胚胎303枚,301枚囊胚活检成功,成功诊断胚胎287个,诊断成功率为95.3%(287/301),获得可供移植的整倍体胚胎共85个,其中18个周期无可用胚胎,周期取消率19.5%。平衡易位组获得整倍体胚胎67个,罗氏易位组获得整倍体胚胎18个,PGT后胚胎着床率分别为89.3%(42/47)和88.8%(8/9),早期流产率分别为25.5%(12/47)和22.2%(2/9),继续妊娠率+活产率分别为63.8%(30/47)和66.6%(6/9),产前诊断结果与PGT结果一致。结论基于NGS的PGT可准确的对胚胎进行染色体病诊断,避免反复流产和非意愿性终止妊娠,并获得理想的临床妊娠率。     

Development and validation of a sample sparing strategy for HLA typing utilizing next generation sequencing

We report the development of a general methodology to genotype HLA class I and class II loci. A Whole Genome Amplification (WGA) step was used as a sample sparing methodology. HLA typing data could be obtained with as few as 300 cells, underlining the usefulness of the methodology for studies for which limited cells are available. The next generation sequencing platform was validated using a panel of cell lines from the International Histocompatibility Working Group (IHWG) for HLA-A, -B, and -C. Concordance with the known, previously determined HLA types was 99%. We next developed a panel of primers to allow HLA typing of alpha and beta chains of the HLA DQ and DP loci and the beta chain of the DRB1 locus. For the beta chain genes, we employed a novel strategy using primers in the intron regions surrounding exon 2, and the introns surrounding exons 3 through 4 (DRB1) or 5 (DQB1 and DPB1). Concordance with previously determined HLA Class II types was also 99%. To increase throughput and decrease cost, we developed strategies combining multiple loci from each donor. Multiplexing of 96 samples per run resulted in increases in throughput of approximately 8-fold. The pipeline developed for this analysis (HLATyphon) is available for download at https://github.com/LJI-Bioinformatics/HLATyphon.     

The use of next generation sequencing in the diagnosis and typing of respiratory infections

BackgroundMolecular assays are the gold standard methods used to diagnose viral respiratory pathogens. Pitfalls associated with this technique include limits to the number of targeted pathogens, the requirement for continuous monitoring to ensure sensitivity/specificity is maintained and the need to evolve to include emerging pathogens. Introducing target independent next generation sequencing (NGS) could resolve these issues and revolutionise respiratory viral diagnostics.ObjectivesTo compare the sensitivity and specificity of target independent NGS against the current standard diagnostic test.Study designDiagnostic RT-PCR of clinical samples was carried out in parallel with target independent NGS. NGS sequences were analyzed to determine the proportion with viral origin and consensus sequences were used to establish viral genotypes and serotypes where applicable.Results89 nasopharyngeal swabs were tested. A viral pathogen was detected in 43% of samples by NGS and 54% by RT-PCR. All NGS viral detections were confirmed by RT-PCR.ConclusionsTarget independent NGS can detect viral pathogens in clinical samples. Where viruses were detected by RT-PCR alone the Ct value was higher than those detected by both assays, suggesting an NGS detection cut-off – Ct = 32. The sensitivity and specificity of NGS compared with RT-PCR was 78% and 80% respectively. This is lower than current diagnostic assays but NGS provided full genome sequences in some cases, allowing determination of viral subtype and serotype. Sequencing technology is improving rapidly and it is likely that within a short period of time sequencing depth will increase in-turn improving test sensitivity.     

Recommendations for next generation sequencing data reanalysis of unsolved cases with suspected Mendelian disorders: A systematic review and meta-analysis

PurposeThe study aimed to determine the diagnostic yield, optimal timing, and methodology of next generation sequencing data reanalysis in suspected Mendelian disorders.MethodsWe conducted a systematic review and meta-analysis of studies that conducted data reanalysis in patients with suspected Mendelian disorders. Random effects model was used to pool the estimated outcome with subgroup analysis stratified by timing, sequencing methodology, sample size, segregation, use of research validation, and artificial intelligence (AI) variant curation tools.ResultsA search of PubMed, Embase, Scopus, and Web of Science between 2007 and 2021 yielded 9327 articles, of which 29 were selected. Significant heterogeneity was noted between studies. Reanalysis had an overall diagnostic yield of 0.10 (95% CI = 0.06-0.13). Literature updates accounted for most new diagnoses. Diagnostic yield was higher after 24 months, although this was not statistically significant. Increased diagnoses were obtained with research validation and data sharing. AI-based tools did not adversely affect reanalysis diagnostic rate.ConclusionNext generation sequencing data reanalysis can improve diagnostic yield. Owing to the heterogeneity of the studies, the optimal time to reanalysis and the impact of AI-based tools could not be determined with confidence. We propose standardized guidelines for future studies to reduce heterogeneity and improve the quality of the conclusions.     

Clinical utility of a targeted next generation sequencing panel in severe and pediatric onset Mendelian diseases

Next generation sequencing has provided great advancements in genetic diagnosis of Mendalian disorders. Simultaneous sequencing of many genes has become increasingly cheaper and faster. Recently, a number of gene panels have been established for the diagnosis of specific disease groups. The aim of this study is to evaluate the utility of an inherited disease panel in pediatric onset Mendelian diseases. Two hundred and seventeen probands and 10 carriers molecularly analyzed using a TruSight Inherited Disease^® Panel which included 552 genes responsible for pediatric onset Mendelian disorders, were enrolled in the study. The clinical phenotype, sequencing data, pretest and posttest diagnoses were evaluated. The patients in the study were classified into two groups. Group 1 (n:209) included the patients having a clinical diagnosis prior to molecular analysis. Group 2 (n:18) included the patients undiagnosed clinically prior to molecular analysis. Targeted panel provided a molecular diagnosis in 37% (84 of 227 cases) of all cases. The molecular diagnostic rate was 40.2% in patients with a specific prior clinical diagnosis. However, in patients having no primary clinical diagnosis no pathogenic variants were found. In 14 patients, a molecular diagnosis differing from the established clinical diagnosis was made. In conclusion, a targeted panel covering a high number of genes responsible for broad phenotypic spectrum can provide improved levels of diagnosis in patients with pediatric onset Mendelian diseases. A careful clinical evaluation of patients prior to the application of a next generation sequencing method leads to the best alternative approach for a conclusive molecular diagnosis.     

Wrestling with the repertoire: The promise and perils of next generation sequencing for antigen receptors

Next generation sequencing technologies are revolutionizing the study of immune repertoires. These methods provide a previously unimaginable amount of sequence data, unfortunately accompanied by numerous challenges associated with error correction and interpretation that remain to be solved. For antigen receptors, these challenges will require dedicated solutions beyond those developed for genome sequencing, which may differ depending on the sequencing technology used and the purpose of the experiment. Many investigators are developing such methods, based on different sequencing platforms, but critical details of protocol and performance are proprietary. The field will move forward when these methods are shared and standardized, and when the accuracy, sensitivity and reproducibility of various sequencing, and analytic methods are evaluated using standardized samples in comparative experiments.