NGS技术检测自然流产胚胎或绒毛染色体非整倍体及拷贝数变异的研究

目的探索新一代测序技术(NGS)在检测自然流产胚胎或绒毛组织染色体非整倍体和拷贝数变异(CNV)应用中的价值。方法选择20例自然流产患者的绒毛进行染色体核型分析,同时应用NGS技术进行染色体非整倍体和拷贝数变异的检测,并以染色体核型分析结果为"金标准"进行NGS方法的评估。后于2013年共收集1074例自然流产胚胎或绒毛组织,应用NGS技术完成染色体非整倍体和CNV的检测,并对检测结果进行分析。结果 20例自然流产样本的NGS结果与核型分析结果对比,检测灵敏度和特异性均为100%。临床检测的1074例流产组织样本中,42例样本DNA不符合质控标准,实际完成检测1032例。1032例组织样本中阳性445例(43.12%),其中非整倍体369例(82.92%),以16、X、22、21、15、18号染色体高发;CNV共76例(17.08%),阳性样本集中发生在8-12w。阴性587例(56.88%)。根据孕妇年龄将样本分为三组比较,差异有统计学意义(P0.01)。结论 NGS技术用于检测流产组织的非整倍体和拷贝数变异具有较高的灵敏度和特异性,是适用于临床的有效检测方法。     

基于游离DNA的纳米孔测序在一例肺炎病例病原识别中的应用

目的探讨游离DNA测序在呼吸道感染病原诊断中的应用价值。方法对一例肺炎病例的支气管肺泡灌洗液和血液样本提取游离DNA, 同时进行纳米孔测序和二代测序。结果纳米孔测序从肺泡灌洗液样本中检测到132条1型单纯疱疹病毒(herpes simplex virus type 1, HSV-1)序列, 但从血液样本中未检出HSV-1序列。二代测序从两种样本中均检出HSV-1序列, 产生的病毒序列数与测序深度高于纳米孔测序。实时定量PCR反应和血清学检测证实了该诊断。纳米孔测序与二代测序数据混合组装后获得了HSV-1全长序列, 系统进化分析表明该病毒与美国分离的毒株亲缘关系最近。结论纳米孔测序技术可在更短时间内检测出病原, 有助于感染性疾病病原的快速诊断。     

应用高通量测序检测妇科恶性肿瘤患者血浆游离DNA的临床效果

目的:分析高通量测序在妇科恶性肿瘤患者血浆游离DNA检测中的应用.方法:收集中山市博爱医院2019年-2020年12月50例血液样本,采用高通量测序技术对其进行测序分析,比较高通量测序技术的准确性及敏感性.结果:对50例血液样本进行分析,检出其中5例确诊为妇科恶性肿瘤患者,4例为子宫内膜样腺癌,1例为卵巢癌,45例为非恶性肿瘤患者.高通量测序结果显示,仅卵巢癌患者发现多条染色体大片段拷贝数变异(copy number variation,CNV),而其余病例未发现染色体数目异常.以最终诊断结果为标准,高通量测序对恶性妇科肿瘤诊断的灵敏度为20.00％,特异度为100.00％.结论:高通量测序技术可检测出卵巢癌患者血浆中的肿瘤细胞游离DNA(cell-free circulating tumor DNA,ctDNA),但子宫内膜癌患者在本次实验中未检测出阳性结果.     

全基因组测序在环境样本新型冠状病毒检测中的应用

目的:通过对环境样本进行全基因组测序,为全基因组测序在检测环境标本中SARS-CoV-2的应用提供实践基础,为新冠肺炎疫情的溯源和防控工作提供科学依据.方法:采用新冠病毒全基因组靶向扩增结合Illumina二代测序的技术对5例SARS-CoV-2核酸阳性环境样本进行全基因组测序,应用多种分析软件及在线病毒变异分析平台,...     

杂合型单核苷酸多态性定量焦测序测定法用于唐氏综合征快速诊断的探索

目的 通过筛查人21号染色体上的杂合型单核苷酸多态性(single nucleotide polymorphism,SNP)位点,初步建立基于定量焦磷酸测序反应的唐氏综合征分子诊断方法.方法 基于改进引物设计的等位基因特异性扩增法,在21号染色体上选取7个SNP位点进行杂合子筛选,通过焦测序法检测SNP中两个等位基因型的比值,并作为诊断唐氏综合征的依据.结果 通过对84名正常中国人基因组样本进行筛查,得到6个杂合率较高的SNP位点.6个SNP位点的杂合子覆盖率为92.9%.应用这6个SNP位点对10例唐氏综合征患者的样本进行了焦测序定量检测,结果表明10例患者样本中有9例样本的两个基因型比值为2:1或1:2,1例未检出杂合子,检出率为90%.结论 初步建立了一种唐氏综合征辅助诊断方法,具有成本低、操作简单、快速和结果明了的优点,可以有效地快速诊断唐氏综合征,为今后进一步完善该方法,并将其用于临床诊断奠定了基础.     

杂合型单核苷酸多态性定量焦测序测定法用于唐氏综合征快速诊断的探索

目的 通过筛查人21号染色体上的杂合型单核苷酸多态性(single nucleotide polymorphism,SNP)位点,初步建立基于定量焦磷酸测序反应的唐氏综合征分子诊断方法.方法 基于改进引物设计的等位基因特异性扩增法,在21号染色体上选取7个SNP位点进行杂合子筛选,通过焦测序法检测SNP中两个等位基因型的比值,并作为诊断唐氏综合征的依据.结果 通过对84名正常中国人基因组样本进行筛查,得到6个杂合率较高的SNP位点.6个SNP位点的杂合子覆盖率为92.9%.应用这6个SNP位点对10例唐氏综合征患者的样本进行了焦测序定量检测,结果表明10例患者样本中有9例样本的两个基因型比值为2:1或1:2,1例未检出杂合子,检出率为90%.结论 初步建立了一种唐氏综合征辅助诊断方法,具有成本低、操作简单、快速和结果明了的优点,可以有效地快速诊断唐氏综合征,为今后进一步完善该方法,并将其用于临床诊断奠定了基础.     

杂合型单核苷酸多态性定量焦测序测定法用于唐氏综合征快速诊断的探索

目的 通过筛查人21号染色体上的杂合型单核苷酸多态性(single nucleotide polymorphism,SNP)位点,初步建立基于定量焦磷酸测序反应的唐氏综合征分子诊断方法.方法 基于改进引物设计的等位基因特异性扩增法,在21号染色体上选取7个SNP位点进行杂合子筛选,通过焦测序法检测SNP中两个等位基因型的比值,并作为诊断唐氏综合征的依据.结果 通过对84名正常中国人基因组样本进行筛查,得到6个杂合率较高的SNP位点.6个SNP位点的杂合子覆盖率为92.9%.应用这6个SNP位点对10例唐氏综合征患者的样本进行了焦测序定量检测,结果表明10例患者样本中有9例样本的两个基因型比值为2:1或1:2,1例未检出杂合子,检出率为90%.结论 初步建立了一种唐氏综合征辅助诊断方法,具有成本低、操作简单、快速和结果明了的优点,可以有效地快速诊断唐氏综合征,为今后进一步完善该方法,并将其用于临床诊断奠定了基础.     

杂合型单核苷酸多态性定量焦测序测定法用于唐氏综合征快速诊断的探索

目的 通过筛查人21号染色体上的杂合型单核苷酸多态性(single nucleotide polymorphism,SNP)位点,初步建立基于定量焦磷酸测序反应的唐氏综合征分子诊断方法.方法 基于改进引物设计的等位基因特异性扩增法,在21号染色体上选取7个SNP位点进行杂合子筛选,通过焦测序法检测SNP中两个等位基因型的比值,并作为诊断唐氏综合征的依据.结果 通过对84名正常中国人基因组样本进行筛查,得到6个杂合率较高的SNP位点.6个SNP位点的杂合子覆盖率为92.9%.应用这6个SNP位点对10例唐氏综合征患者的样本进行了焦测序定量检测,结果表明10例患者样本中有9例样本的两个基因型比值为2:1或1:2,1例未检出杂合子,检出率为90%.结论 初步建立了一种唐氏综合征辅助诊断方法,具有成本低、操作简单、快速和结果明了的优点,可以有效地快速诊断唐氏综合征,为今后进一步完善该方法,并将其用于临床诊断奠定了基础.     

杂合型单核苷酸多态性定量焦测序测定法用于唐氏综合征快速诊断的探索

目的 通过筛查人21号染色体上的杂合型单核苷酸多态性(single nucleotide polymorphism,SNP)位点,初步建立基于定量焦磷酸测序反应的唐氏综合征分子诊断方法.方法 基于改进引物设计的等位基因特异性扩增法,在21号染色体上选取7个SNP位点进行杂合子筛选,通过焦测序法检测SNP中两个等位基因型的比值,并作为诊断唐氏综合征的依据.结果 通过对84名正常中国人基因组样本进行筛查,得到6个杂合率较高的SNP位点.6个SNP位点的杂合子覆盖率为92.9%.应用这6个SNP位点对10例唐氏综合征患者的样本进行了焦测序定量检测,结果表明10例患者样本中有9例样本的两个基因型比值为2:1或1:2,1例未检出杂合子,检出率为90%.结论 初步建立了一种唐氏综合征辅助诊断方法,具有成本低、操作简单、快速和结果明了的优点,可以有效地快速诊断唐氏综合征,为今后进一步完善该方法,并将其用于临床诊断奠定了基础.     

杂合型单核苷酸多态性定量焦测序测定法用于唐氏综合征快速诊断的探索

目的 通过筛查人21号染色体上的杂合型单核苷酸多态性(single nucleotide polymorphism,SNP)位点,初步建立基于定量焦磷酸测序反应的唐氏综合征分子诊断方法.方法 基于改进引物设计的等位基因特异性扩增法,在21号染色体上选取7个SNP位点进行杂合子筛选,通过焦测序法检测SNP中两个等位基因型的比值,并作为诊断唐氏综合征的依据.结果 通过对84名正常中国人基因组样本进行筛查,得到6个杂合率较高的SNP位点.6个SNP位点的杂合子覆盖率为92.9%.应用这6个SNP位点对10例唐氏综合征患者的样本进行了焦测序定量检测,结果表明10例患者样本中有9例样本的两个基因型比值为2:1或1:2,1例未检出杂合子,检出率为90%.结论 初步建立了一种唐氏综合征辅助诊断方法,具有成本低、操作简单、快速和结果明了的优点,可以有效地快速诊断唐氏综合征,为今后进一步完善该方法,并将其用于临床诊断奠定了基础.     

Comprehensive Genome Profiling of Single Sperm Cells by Multiple Annealing and Looping‐Based Amplification Cycles and Next‐Generation Sequencing from Carriers of Robertsonian Translocation

Robertsonian translocation (RT) is a common cause for male infertility, recurrent pregnancy loss, and birth defects. Studying meiotic recombination in RT‐carrier patients helps decipher the mechanism and improve the clinical management of infertility and birth defects caused by RT. Here we present a new method to study spermatogenesis on a single‐gamete basis from two RT carriers. By using a combined single‐cell whole‐genome amplification and sequencing protocol, we comprehensively profiled the chromosomal copy number of 88 single sperms from two RT‐carrier patients. With the profiled information, chromosomal aberrations were identified on a whole‐genome, per‐sperm basis. We found that the previously reported interchromosomal effect might not exist with RT carriers. It is suggested that single‐cell genome sequencing enables comprehensive chromosomal aneuploidy screening and provides a powerful tool for studying gamete generation from patients carrying chromosomal diseases.     

2433例孕妇血浆胎儿游离DNA无创性产前非整倍性检测结果分析

目的探讨应用高通量测序技术对孕妇血浆胎儿游离DNA进行无创性胎儿染色体非整倍性检测的准确性。方法选择2011年10月至2013年9月于佛山市妇幼保健院行无创性非整倍性产前基因检测的2433例名孕妇,孕周12-24w,均为单胎,年龄21-41岁。对无创性非整倍性筛查高风险的孕妇行羊膜腔穿刺或脐静脉血穿刺,行常规染色体核型分析。对筛查结果低风险者行电话随访其胎儿出生后情况,统计分析无创性产前非整倍性检测的准确性。结果2433例孕妇中,母体血浆胎儿游离DNA高通量测序技术检测出42例胎儿染色体非整倍性高风险。其中33例通过羊膜腔穿刺或脐静脉血穿刺对比分析,27例21三体高风险者行有创性产前诊断,26例为47,XN,＋21,l例为46,XN。2例18三体高风险者行进一步确诊,其中1例为47,XN,＋18,1例为46,XN。2例i3三体高风险孕妇结果均与穿刺结果为46,XN。1例XO高风险孕妇与穿刺结果为45X[25]／46,XX[25]。1例XXY高风险孕妇结果为47,XXY。孕妇血浆中游离胎儿DNA检测结果阴性者2391例,经电话随访有效者2145例,截止至2014年4月30日,已出生的新生儿均未发现唐氏综合征患儿。无创性非整倍体检测对常见染色体非整倍体的检出率100％,灵敏废100％,特异度99.8,假阳性率0.33％,假阴性率为0,阳性预测值87.9％。结论应用高通量测序技术在染色体非整倍性无创性检测具有很高的灵敏性,假阳性率很低,在胎儿染色体非整倍性疾病的产前检测中具有广泛的应用前景。     

Close correlation of copy number aberrations detected by next‐generation sequencing with results from routine cytogenetics in acute myeloid leukemia

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor‐specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well‐defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (<33%) was limiting for CNA detection. We detected CNAs consistent with cytogenetics in 83% of AML samples including highly correlated clone size estimation (R = 0.85), while six out of 65 cytogenetically normal AML samples exhibited CNAs apparently missed by routine cytogenetics. Overall, our results show that high throughput targeted sequencing data can be reliably used to detect copy number changes in the dominant AML clone. © 2016 Wiley Periodicals, Inc.     

A Robust Approach for Blind Detection of Balanced Chromosomal Rearrangements with Whole‐Genome Low‐Coverage Sequencing

Balanced chromosomal rearrangement (or balanced chromosome abnormality, BCA) is a common chromosomal structural variation. Next‐generation sequencing has been reported to detect BCA‐associated breakpoints with the aid of karyotyping. However, the complications associated with this approach and the requirement for cytogenetics information has limited its application. Here, we provide a whole‐genome low‐coverage sequencing approach to detect BCA events independent of knowing the affected regions and with low false positives. First, six samples containing BCAs were used to establish a detection protocol and assess the efficacy of different library construction approaches. By clustering anomalous read pairs and filtering out the false‐positive results with a control cohort and the concomitant mapping information, we could directly detect BCA events for each sample. Through optimizing the read depth, BCAs in all samples could be blindly detected with only 120 million read pairs per sample for data from a small‐insert library and 30 million per sample for data from nonsize‐selected mate‐pair library. This approach was further validated using another 13 samples that contained BCAs. Our approach advances the application of high‐throughput whole‐genome low‐coverage analysis for robust BCA detection—especially for clinical samples—without the need for karyotyping.     

染色体微阵列分析技术(CMA)在产前诊断中的应用研究

目的本研究对410例羊水标本同时行传统染色体核型分析及CMA检测的结果进行对比分析,探讨CMA在产前诊断中应用的优势与不足及CMA结果的解读和咨询难点。方法 2017年9月~2018年9月,共410例孕妇在怀化市产前诊断中心进行产前诊断,同时行传统染色体核型分析及CMA检测,对两种检测方法结果进行比对分析。结果本研究410例标本中传统染色体核型分析检出的24例染色体非整倍体异常及5例胎儿染色体不平衡变异,CMA均能检出,在传统染色体核型分析正常的病例中CMA检测出了6例杂合性缺失、48例临床意义不明的拷贝数变异55个(检出率13.4%(55/410)),7例致病性异常,疾病检出率提高了1.7%,结论与传统染色体核型分析比较,CMA技术不仅能检测出非整倍体及不平衡染色体变异,且具有更高的分辨率,能检测具有临床意义的拷贝数变异,CMA技术在产前诊断中的作用将会更加重要。     

Intra-host non-synonymous diversity at a neutralizing antibody epitope of SARS-CoV-2 spike protein N-terminal domain

ObjectivesSARS-CoV-2 has evolved rapidly into several genetic clusters. However, data on mutations during the course of infection are scarce. This study aims to determine viral genome diversity in serial samples of COVID-19 patients.MethodsTargeted deep sequencing of the spike gene was performed on serial respiratory specimens from COVID-19 patients using nanopore and Illumina sequencing. Sanger sequencing was then performed to confirm the single nucleotide polymorphisms.ResultsA total of 28 serial respiratory specimens from 12 patients were successfully sequenced using nanopore and Illumina sequencing. A 75-year-old patient with severe disease had a mutation, G22017T, identified in the second specimen. The frequency of G22017T increased from ≤5% (nanopore: 3.8%; Illumina: 5%) from the first respiratory tract specimen (sputum) to ≥60% (nanopore: 67.7%; Illumina: 60.4%) in the second specimen (saliva; collected 2 days after the first specimen). The difference in G22017T frequency was also confirmed by Sanger sequencing. G22017T corresponds to W152L amino acid mutation in the spike protein which was only found in <0.03% of the sequences deposited into a public database. Spike amino acid residue 152 is located within the N-terminal domain, which mediates the binding of a neutralizing antibody.DiscussionA spike protein amino acid mutation W152L located within a neutralizing epitope has appeared naturally in a patient. Our study demonstrated that monitoring of serial specimens is important in identifying hotspots of mutations, especially those occurring at neutralizing epitopes which may affect the therapeutic efficacy of monoclonal antibodies.     

Chromosomal abnormalities among 246 fetuses with pleural effusions detected on prenatal ultrasound examination: factors associated with an increased risk of aneuploidy.

PURPOSE: To determine the prevalence of chromosomal abnormalities in fetuses with prenatally diagnosed pleural effusions and to identify factors associated with an increased risk of aneuploidy. METHODS: A retrospective analysis of the Genzyme Genetics database was performed for samples submitted from October 1994 to April 2003 with an indication of fetal pleural effusion. RESULTS: There were 246 samples in which pleural effusion was identified as an indication for prenatal chromosome analysis. Ninety-four were from fetuses with isolated pleural effusions and 152 had other abnormalities in addition to pleural effusion. The prevalence of chromosome abnormalities was 35.4% (95% confidence interval, 29.2-41.4%). Among the eight first trimester samples, the aneuploidy rate was 63%. Pleural effusion cases associated with additional sonographic findings had a significantly higher aneuploidy rate than the isolated pleural effusion cases (50% vs. 12%, P < 0.001). CONCLUSIONS: Chromosome analysis is warranted after the prenatal detection of a fetal pleural effusion. The risk of aneuploidy is greater with first trimester detection and is significantly increased in the presence of other associated anomalies.     

Performance of prenatal screening by non-invasive cell-free fetal DNA testing for women with various indicationsCSCD

Objective: To assess the performance of non-invasive prenatal testing (NIPT) based on massive parallel sequencing. Methods: A total of 10 275 maternal blood samples were collected. Fetal chromosomal aneuploides were subjected to low coverage whole genome sequencing. Patients with high risks received further prenatal diagnosis. The outcome of all patients were followed up. Results: High-throughput sequencing detected 72 pregnancies with fetal autosomal chromosomal aneuploidy, including 57 cases of trisomy 21, 14 cases of trisomy 18, and 1 case of trisomy 13. The positive predictive value for trisomies 21 and 18 were 98.25% and 91.67%, respectively. Comparing its performance in intermediate or high risk pregnancies, advanced maternal age pregnancies and volunteering to test pregnancies, the positive predictive value were 100%, 95%, 90% and 50%, respectively. The follow up result was only 1 case of 21 trisomy false negative with high risk. For the 56 cases of trisomy 21, the high risk group accounted for 55%, advanced maternal age accounted for 29%, the intermediate risk referred to 14%, the volunteering to test group accounted for 2%. Conclusion: The performance of NIPT for trisomies 21, 18 and 13 was satisfactory. The method can be used for women with advanced gestational age. NIPT has offered an ideal secondary screening method for those with an intermediate or high risk, and can reduce the rate of birth defects. © 2018 West China University of Medical Sciences. All rights reserved.     

MLPA技术在检测胎儿非整倍体异常中的应用

目的探讨多重探针连接依赖式扩增（multiplex ligation—dependent probe amplification,MLPA）在快速检测胎儿染色体非整倍体异常中的应用价值。方法344例产前诊断标本同时进行MLPA检测及核型分析,所有样本进行MLPA获得的扩增产物信息经Coffalyserv9．4软件（Holland—MRC公司）进行定量分析,观察样本DNA拷贝数的变化,并将所得结果与染色体核型分析结果进行比较,计算其检测的敏感度、特异度及阳性预测值。结果MLPA分析在标本接收后24h内即可得出结果,共检出染色体倍体异常产前诊断标本5例,包括唐氏综合征（Downsyndrome,47,＋21）6例、爱德华氏综合征（Edwardssyndrome,47,＋18）l例。MLPA检测24h报告结果临床符合率为97．7％。结论MLPA检测21、18、13、X、Y等染色体非整倍体疾病时,与核型分析相比较,MLPA是一种快速、高效的分析非整倍体的产前诊断的方法,具有临床应用价值。