高通量测序平台测定混合病毒样品基因组序列

目的 建立RNA病毒基因组的从头测序方法,验证高通量测序平台同时测定不同RNA病毒基因组序列的能力。方法 选取日常监测工作中分离的一株EV71毒株和一株季节性流感病毒,提取病毒RNA,制备测序文库,按标准步骤在GS-Junior完成测序反应,分析数据。结果 共得到超过14万序列片段,总计46.9 M碱基,平均读长334 bp。初步拼接可以得到914个片段(contig), 而以相应参考毒株基因组为标准,一次反应得到EV71全长基因组的99.84%及流感病毒的96.7%。结论 高通量测序仪能够同时快速、准确地从头测定多株RNA病毒基因组序列,适合应用于多个病原生物学领域。     

高通量测序技术检测肥厚型心肌病致病基因研究

目的:利用高通量测序技术对肥厚型心肌病(HCM)患者进行基因筛查,并与临床表型相对照,以期对临床诊治HCM提供参考依据。方法:对19例门诊HCM患者进行高通量测序,检测其突变类型并收集临床资料。结果:19例患者中有9例发现基因突变,除2例患者为单磷酸腺苷(AMP)激活蛋白激酶γ2调节亚基(PRKAG2)突变外,大部分基因突变为肌节蛋白基因突变。此外,检测出2例双基因突变患者,涉及PRKAG2与肌球蛋白结合蛋白C(MYBPC3)、MYBPC3与β-重链肌球蛋白(MYH7)双基因突变。基因突变的检出率为47%。MYH7基因突变患者发病年龄相对较轻,心肌肥厚程度较重,2例PRKAG2基因突变患者均有不同程度的心肌肥厚和传导阻滞的表现。结论:高通量测序技术准确、高效,对于HCM致病基因的检测阳性率高,与临床表型相对照,对于HCM的发生、发展以及转归提供了有利的依据。     

一株临床耐药菌高通量测序数据的深度挖掘

目的通过分析一株临床耐药菌高通量测序的数据,探讨从二代测序原始数据中挖掘提取有用信息的流程。方法综合利用主流生物信息学软件,筛选可能和耐药有关的突变位点。结果通过对基因组测序数据的处理和从头组装,得到该菌株基因组的基本特征、进化关系及可能和抗药性有关的突变位点。结论随着高通量测序技术的发展,临床分离出的致病菌的测序已经成了常规的研究技术。通过对测序数据的深度挖掘,将为临床研究致病菌的致病和耐药机制提供更有价值的线索。     

基于高通量测序的粉尘螨体内细菌多样性研究

目的 探讨粉尘螨（Dermatophagoides farinae）体内细菌多样性。方法 取实验室人工饲养的粉尘螨,采用Illumina PE250高通量测序平台扩增测序细菌16S核糖体RNA(16S ribosomal RNA, 16S rRNA)基因V4区,获得的原始序列经质控过滤后得到有效序列。采用Usearch软件、Silva数据库、Mothur软件对有效序列进行操作分类单元（operational taxonomic units,OTU）聚类并分析细菌群落组成与alpha多样性指数。结果 共获得187 616条有效序列,根据序列相似性> 97%进行聚类,得到469个OTUs。OTU注释结果表明,粉尘螨体内细菌分属于26个门、43个纲、100个目、167个科、284个属。在门分类阶元水平,粉尘螨体内细菌主要注释到变形菌门、厚壁菌门、拟杆菌门、放线菌门和酸杆菌门,其中变形菌门为优势菌门;在属分类阶元水平,罗尔斯通氏菌属、立克次体目未确定属、葡萄球菌属和鞘氨醇单胞菌属为优势菌属。在非优势菌属中,发现沃尔巴克氏体。结论 粉尘螨体内细菌群落组成具有较高多样性,个体间细菌群落多样性...     

高通量测序技术在临床病毒学领域的应用北大核心CSCD

本文总结了近几年高通量测序平台的发展现状,比较了不同测序平台的性能及特点。同时还对目前高通量测序平台在近两年临床病毒学领域的应用,包括病原学诊断、分子流行病学、宏基因组学和临床治疗转归等几个方面,逐一举例进行了阐述。籍此加深专业人员对高通量测序平台的认识,促进该技术在临床病毒学的转化与应用。     

日本血吸虫虫卵分泌物小 RNA的高通量测序

目的 通过对日本血吸虫虫卵分泌物（ ｅｇｇ ｓｅｃｒｅｔｅｄ ｐｒｏｄｕｃｔｓ,ＥＳＰ） 的小 ＲＮＡ 进行高通量测序,分析、鉴定已知 ｍｉＲＮＡ 的表达水平。 方法 提取日本血吸虫虫卵 ＥＳＰ ｍｉＲＮＡ 进行高通量测序;通过对数据分析包括统计长度分布与比对注释,鉴定已知 ｍｉＲＮＡ 的表达水平。 结果 将构建文库中日本血吸虫虫卵ＥＳＰ 表达的 ｍｉＲＮＡ 与最新 ｍｉＲＢａｓｅ 数据库中的 ｍｉＲＮＡ 比对,发现在鉴定的 １２ 个 ｍｉＲＮＡｓ 中,ｓｊａ － ｍｉＲ －３４８８ 为表达量最高的 ｍｉＲＮＡ,其次为 ｓｊａ － ｍｉＲ － ３６ － ３ｐ 和 ｓｊａ － ｍｉＲ － ７１ａ,上述三种 ｍｉＲＮＡ 表达量占比９１ ８％ 。 结论 日本血吸虫虫卵 ＥＳＰ 三个 ｍｉＲＮＡｓ（ ｓｊａ － ｍｉＲ － ３４８８、ｓｊａ － ｍｉＲ － ３６ － ３ｐ 和 ｓｊａ － ｍｉＲ － ７１ａ）的鉴定为下一步探寻血吸虫病诊疗新靶标奠定了基础。     

羊水细胞高通量测序与染色体核型分析在产前诊断中的应用研究

目的通过高通量测序技术(即下一代测序技术,next generation sequencing,NGS)检测孕妇羊水细胞DNA,与染色体核型分析进行对比,探索NGS在羊水细胞产前诊断中的应用价值。方法选取孕龄在18~24周之间的高龄妊娠、唐氏综合征生化筛查高风险和(或)彩超显示胎儿异常并同意产前诊断的孕妇101例,抽取孕妇羊水,提取羊水细胞DNA,制备测序文库,应用Ion Proton测序仪检测,所得的DNA序列与人类DNA参考数据库比对并作统计分析,并与同一样本染色体核型分析进行对照分析。结果 101例羊水样本处理后经NGS技术检测判定2例染色体数目异常,37例染色体片段缺失/重复;羊水细胞培养检出2例染色体数目异常,2例9号染色体臂间倒位,2例多态性。结论利用高通量测序技术检测孕妇羊水中DNA诊断胎儿染色体数目异常,其特异性与染色体核型分析技术具有较高的一致性,并可检测出缺失/重复。染色体核型分析技术与高通量测序技术相结合在检测出生缺陷上具有较好的临床实际应用价值,并可进一步展开对疾病候选基因的研究。     

高通量测序对脓毒症病原学诊断及病原菌耐药性预测的应用价值

脓毒症是机体对感染反应失调所致的危及生命的器官功能障碍,是急危重症医学面临的重要疾病,也是临床亟待解决的难题。明确病原学后尽早针对性抗感染治疗可降低病死率,是治疗脓毒症的关键。目前的病原学检测技术在及时性和准确性等方面仍存在欠缺。高通量测序技术在诊断病原微生物方面,具有检测种类多、快速、精准等特点,且近年对于监测病原学及耐药性方面有特殊优势,并逐步应用于临床。该文就高通量测序对脓毒症病原学诊断及病原菌耐药性预测的应用价值进行综述。     

运用高通量RNA测序探讨绝经后骨质疏松与免疫功能的关系

目的探讨绝经后骨质疏松与免疫功能的关系。方法选取6例符合要求的绝经后病人,3例骨质疏松患者为实验组,3例非骨质疏松者为对照组。双能X线吸收仪测定骨密度。空腹抽取肘静脉血适量,提取外周血单个核细胞RNA。Nanodrop分光光度计及琼脂糖电泳对RNA进行质检,构建RNA文库。用Illumina Hiseq2000平台测序,对数据进行统计,寻找差异表达基因,并且对这些差异表达基因进行功能富集分析。结果根据差异分析P0.05阈值筛选两组的差异基因,这些差异表达基因功能富集显示,大多集中在与免疫相关的信号通路上,如防御反应(IL-15,IL-1 RAP,TNFAIP6,TNFRSF4,IL-2RA,IL-8等),免疫反应(CD8A,CD8B,IL-15,TNFRSF13C CD79B,CD14等),炎症反应(IL-15,CD24,TLR10,TNFAIP6等),白细胞活化(IL-8,CD8A,CD8B等)和淋巴细胞活化(CD3G,CD8A,CD8B,THEMIS,KIR2DS4,KIR3DL1等)。结论绝经后骨质疏松的发生与患者的免疫功能改变有一定的关系。     

Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.     

基于高通量测序分析入境羊毛微生物群落结构特征

目的 通过高通量测序的方法了解来自5个国家5批入境羊毛表面携带的致病菌和微生物群落结构特征,为国境卫生以及传染病预防提供有效的数据支撑。方法 通过浸泡搅拌震荡的方法收集5批羊毛样本表面的总细菌并提取DNA,采用Illumina Miseq高通量测序的方法,主要分析比较5批羊毛样本的α-多样性指数、微生物群落结构以及致病菌。结果 α-多样性指数的结果表明5批羊毛表面微生物群落结构的多样性具有明显差异,其中德国样本的多样性最高,法国样本的多样性最低,美国、阿根廷、比利时则介于之间。主成分分析结果表明德国和比利时样本的微生物组成相似度较高且与其他3个国家之间的微生物群落结构组成有明显差异。微生物群落结构的16S绝对定量结果表明在微生物总量绝对丰度层面美国>阿根廷>德国>比利时>法国,阿根廷、德国、比利时3个国家的羊毛表面微生物以变形菌门(Proteobacteria)为主,厚壁菌门(Firmicutes)其次,而美国、法国以厚壁菌门(Firmicutes)为主,变形菌门(Proteobacteria)其次。在种属水平上,5批羊毛样本均检测出鲁氏不动杆菌(Acinetobacter lwoffii)、肠沙门菌(Salmonella enterica)和嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)等临床常见的条件致病菌,其中美国样本中肠沙门菌的丰度最高,比利时样本的肠沙门菌的丰度最低。结论 根据测序的结果得出羊毛表面病原体含量较多且疫情复杂,检测出了肠沙门菌等危害性较大的致病菌,相关部门以及一线从业人员应注意做好生物防护。     

Molecular Analysis of SARS-CoV-2 Lineages in Armenia

The sequencing of SARS-CoV-2 provides essential information on viral evolution, transmission, and epidemiology. In this paper, we performed the whole-genome sequencing of SARS-CoV-2 using nanopore and Illumina sequencing to describe the circulation of the virus lineages in Armenia. The analysis of 145 full genomes identified six clades (19A, 20A, 20B, 20I, 21J, and 21K) and considerable intra-clade PANGO lineage diversity. Phylodynamic and transmission analysis allowed to attribute specific clades as well as infer their importation routes. Thus, the first two waves of positive case increase were caused by the 20B clade, the third peak caused by the 20I (Alpha), while the last two peaks were caused by the 21J (Delta) and 21K (Omicron) variants. The functional analyses of mutations in sequences largely affected epitopes associated with protective HLA loci and did not cause the loss of the signal in PCR tests targeting ORF1ab and N genes as confirmed by RT-PCR. We also compared the performance of nanopore and Illumina short-read sequencing and showed the utility of nanopore sequencing as an efficient and affordable alternative for large-scale molecular epidemiology research. Thus, our paper describes new data on the genomic diversity of SARS-CoV-2 variants in Armenia in the global context of the virus molecular genomic surveillance.     

Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K_d < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have ∼3–8-fold higher affinity and ∼2–4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.     

高通量测序在鸟类肠道微生物中的研究进展

鸟类作为分布广泛、种群数量庞大和高度多样化的物种之一,在病原体传播中发挥了重要作用。本文综述了基于高通量测序技术的3种测序策略(16S rDNA测序、宏基因组测序和宏转录组测序)的特点及其在鸟类肠道菌群多样性、抗生素抗性基因和病原微生物发现中的应用,并对未来发展趋势进行了展望。     

Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles

Individual variation in germline and expressed B-cell immunoglobulin (Ig) repertoires has been associated with aging, disease susceptibility, and differential response to infection and vaccination. Repertoire properties can now be studied at large-scale through next-generation sequencing of rearranged Ig genes. Accurate analysis of these repertoire-sequencing (Rep-Seq) data requires identifying the germline variable (V), diversity (D), and joining (J) gene segments used by each Ig sequence. Current V(D)J assignment methods work by aligning sequences to a database of known germline V(D)J segment alleles. However, existing databases are likely to be incomplete and novel polymorphisms are hard to differentiate from the frequent occurrence of somatic hypermutations in Ig sequences. Here we develop a Tool for Ig Genotype Elucidation via Rep-Seq (TIgGER). TIgGER analyzes mutation patterns in Rep-Seq data to identify novel V segment alleles, and also constructs a personalized germline database containing the specific set of alleles carried by a subject. This information is then used to improve the initial V segment assignments from existing tools, like IMGT/HighV-QUEST. The application of TIgGER to Rep-Seq data from seven subjects identified 11 novel V segment alleles, including at least one in every subject examined. These novel alleles constituted 13% of the total number of unique alleles in these subjects, and impacted 3% of V(D)J segment assignments. These results reinforce the highly polymorphic nature of human Ig V genes, and suggest that many novel alleles remain to be discovered. The integration of TIgGER into Rep-Seq processing pipelines will increase the accuracy of V segment assignments, thus improving B-cell repertoire analyses.The production by B cells of immunoglobulin (Ig) proteins, which are expressed on the cell surface as B-cell receptors and secreted by subsets of B cells as antibodies, is a key component of the adaptive immune system in humans. Through their specific binding to an enormously diverse range of foreign bodies, Ig proteins are able to elicit further immunological response and provide protection. These proteins are assembled in B cells from two pairs of polypeptide chains, termed heavy and light. The antigen-binding portions of these genes are created through the somatic recombination of gene segments, termed variable (V), diversity (D), and joining (J). During the recombination process, one each of the ∼46 V, 23 D, and 6 J gene segments (1) recombine to make the antigen-binding region of the heavy chain; the light chain is created by a similar process, although involving one of two different loci (λ and κ) containing V and J genes only. Over three million different Ig sequences can be created through this V(D)J recombinatorial process alone (2). The potential diversity of these sequences is further expanded to the order of trillions (2) when combined with the random insertion and deletion of nucleotides at the gene segment junctions and with somatic hypermutation (SHM), the latter of which introduces nucleotide changes at a rate of of 10⁻³ per base pair per division (3, 4).Variations in a subject’s germline gene segment alleles and expressed repertoire (i.e., the collection of different Igs circulating in that subject) have been associated with various aspects of immune system and health status. Previous studies have, for example: revealed the association of certain germline genotypes with susceptibility to such diseases as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and multiple sclerosis (MS) (5, 6); found correlations of age with a reduced Ig clonal diversity and less intense response to immune challenge (7, 8); found overly expanded clones in cases of lymphoma (9, 10); and discovered convergent Ig evolution across subjects in response to certain immune challenges (11, 12). These repertoire-sequencing (Rep-Seq) studies have benefitted from improvements in sequencing technologies, which allow for the generation of millions of reads per run (13). Previously, the 454 platform (Roche) was preferred because of its unique ability to generate reads long enough to span the V(D)J rearrangement, although now the MiSeq platform (Illumina) is able to generate paired-end reads also long enough to span the V(D)J rearrangement (13). Rep-Seq use is growing rapidly, even spurring the creation of commercial start-ups to provide researchers and clinical laboratories with sequencing and analysis services (14).Analysis of Rep-Seq data depends critically on the determination of the germline V, D, and J alleles used by each of the Ig sequences, and several methods exist to perform the task of V(D)J germline assignment (2, 15–17). All of these methods essentially involve alignment of sample sequences to a database of germline alleles of all known V, D, and J gene segments. The IMGT (18) database of germline Ig alleles is the most widely used, and the National Center for Biotechnology Information refers to IMGT for its reference genome. However, recent studies have discovered the presence of numerous V segments and alleles not reported in any published databases (6, 19–21), as well as a several novel D and J alleles (19). Some of these V alleles have been incorporated into the IMGT database or alternative databases of germline alleles [such as VBASE2 (22) or the UNSWIg human heavy chain repertoire (23)]. The completeness of the germline V(D)J database may greatly influence downstream analysis results, including clinically relevant decision processes (24), as unreported alleles can skew estimated segment distributions and because novel polymorphisms will appear as recurrent somatic mutations.No automated methods exist for detection of novel V(D)J alleles, and most current Rep-Seq studies simply assume that the current databases are complete. One strategy to search for potential Ig polymorphisms involves identifying, from among the least mutated sequences of each clonal group, V genes that have a high frequency of mutation to a single nucleotide at a given position (10% if the nucleotide is a classical SHM hotspot, 5% otherwise), use a wide variety of D and J alleles, and can be ruled out as not having resulted from a PCR chimera (19). Although application of such filtering-based methods have successfully identified novel alleles (19), the resulting predictions require manual curation, and their sensitivity and specificity have not been evaluated. It is unclear whether this approach can distinguish polymorphic positions from SHM hot-spots, which can be mutated in >40% of sequences (25, 26). Here we present a Tool for Immunoglobulin Genotype Elucidation via Rep-Seq (TIgGER). TIgGER includes a sensitive algorithm for the identification of novel V segment alleles, as well as an Ig genotype-determination step, which it uses to correct germline allele assignments from existing V(D)J assignment tools, like IMGT/HighV-QUEST. Application of TIgGER to Rep-Seq data from seven subjects identified 11 new alleles, demonstrating the importance of incorporating novel allele detection into analysis pipelines. TIgGER is available at clip.med.yale.edu/tigger.     

Prenatal diagnosis of chromosome 18 long arm deletion syndrome by high-throughput sequencing: Two case reports

Rationale:Chromosome 18 long arm deletion syndrome is a group of clinical syndromes caused by partial or total genetic material deletion of the long arm of chromosome 18 (18q), whose clinical manifestations are related to presentation and developmental abnormalities in various aspects such as intelligence, face, and movement. Prenatal diagnosis of this syndrome is challenging because of its low incidence and uncharacteristic prenatal clinical performance. In this paper, 2 cases of partial deletion of 18q found in prenatal amniotic fluid examination by high-throughput sequencing were reported and analyzed.Patient concerns:In patient 1, non-invasive prenatal gene detection at 21 + 2 weeks of gestation suggests a risk of trisomy 18. In patient 2, ultrasound examination at 21 + 2 weeks of gestation revealed a single live fetus, but it was difficult to pinpoint whether the fetus had only 1 umbilical artery to supply blood.Diagnosis and intervention:The 18q deletion syndrome was diagnosed by chromosome karyotype analysis and high-throughput sequencing.Outcomes:The pregnancies were terminated due to the abnormal chromosome.Lesson:This report adds novel variants to the genetic profile of 18q deletion, in order to enrich the genetic data of long arm deletion of 18 chromosomes and provide better services for pre-screening, diagnosis, and genetic counseling for this disease.     

高通量测序技术在寄生虫学中的应用

高通量测序技术具有读长较短和能一次并行对几十万到几百万条DNA分子进行序列测定等特征.与传统Sanger测序技术相比,高通量测序技术具有高通量、高分辨率、低成本等优点,极大地促进了测序技术在生命科学研究中的应用.该文对高通量测序技术及其在寄生虫基因组学、转录组学及蛋白组学方面的应用进行综述,讨论高通量测序技术在寄生虫学应用中的发展前景和存在问题.