基于高通量测序数据的微生物检测算法研究进展

新一代高通量测序技术的发展,推动了多个相关研究领域的发展。国际上许多研究机构正在研究利用高通量测序数据进行微生物检测的算法,目前已有一些基于高通量测序数据的微生物检测算法流程设计成功并公开发布。该文通过调研利用高通量测序数据进行微生物检测的相关文献,研究已发布的基于高通量测序数据的微生物检测算法的功能和实现流程,分析几个有代表性算法的优点和不足。最后,对这些检测算法的设计思路进行总结和分类,提出基于高通量测序数据的微生物检测算法的改进设想。     

2014新一代测序论坛: 从技术到应用

随着近年来第二代测序技术的发明与推广,基因序列测序速度极大提升、成本大幅下降,使得基因组测序的大规模应用成为可能。同时,随着测序深度的提高,全基因组关联分析(genome-wide association study, GWAS)、外显子测序、转录组测序、非编码RNA(如miRNA)测序、甲基化测序等新技术的应用,我们对许多物种的基因组,包括外显子、非编码区、转录组和基因拷贝数变异有了更深入、全面的认识,对生物性状、疾病的解析也取得了重大的突破。     

纳米孔三代测序在HIV/AIDS合并肺部感染者快速病原学鉴定的应用价值探讨

目的通过对人类免疫缺陷病毒/获得性免疫缺陷综合征（Human Immunodeficiency Virus/Acquired Immunedeficiency Syndrome,HIV/AIDS）合并肺部感染者纤维支气管镜肺泡灌洗液样本进行研究,探讨纳米孔三代测序（Oxford Nanopore Technologies）对样本快速病原学鉴定的准确性。方法本研究共纳入2例HIV/AIDS合并肺部感染并留取肺泡灌洗液样本的患者,使用纳米孔三代测序对样本DNA进行测序,提取不同时间点的测序数据进行生物信息学分析,并与常规病原体检查和二代测序（Illumina）等方法进行比较,评价纳米孔三代测序对样本快速病原学鉴定的准确性。结果本研究纳入的患者A常规病原学检查和二代测序的结果均提示铜绿假单胞菌感染,纳米孔三代测序仅需1 h测序数据即发现样本中存在铜绿假单胞菌的基因序列;患者B常规病原学检查和二代测序的结果均提示肺炎克雷伯杆菌,屎肠球菌和结核分枝杆菌混合感染,纳米孔三代测序1 h的测序结果即可检测出肺炎克雷伯杆菌和屎肠球菌的基因序列,6 h的测序结果即可检测出结核分枝杆菌的序列。因此,加上对肺泡灌洗液样本处理、DNA文库构建和生物信息学分析的耗时（合计约2 h）,仅需8 h左右即可检测出上述样本的致病微生物。结论纳米孔三代宏基因组测序可对HIV/AIDS合并肺部感染者进行快速病原微生物鉴定,其准确性与常规病原学检查和Illumina二代测序的结果一致。     

临床骨感染诊断中宏基因组测序的应用及发展

骨感染是临床中严重的感染性疾病.准确的诊断是成功治疗和感染控制的第一步,然而基于传统细菌培养的诊断方法存在诸多不足,往往无法对临床治疗做出及时且准确的指导.随着宏基因组测序技术的不断成熟,成本不断降低,其快速、准确、客观等优点逐渐凸显,近年来将其应用于骨感染诊断中的研究层出不穷,但临床中对宏基因组测序技术的了解和认识尚浅.笔者将对宏基因组测序法的原理,在骨感染诊断中的优势和应用现状,以及发展方向作一综述.     

转录组测序在牧草抗逆基因挖掘方面的应用前景

本文系统阐述了转录组测序技术(RNA-seq)的原理和技术流程。查阅近五年国内外的文献,梳理了植物在干旱、盐胁迫及温度胁迫等逆境下的转录组表达方面的研究情况,发现有关牧草方面的转录组研究相对较少且不够深入。RNA-seq在牧草优良种质资源抗逆基因挖掘方面的应用前景十分广阔。     

基于单细胞测序构建基因共表达网络发现新冠病毒感染标志物

目的 基于新冠病毒感染者的外周血单细胞转录组测序数据,构建基因共表达网络,探究新冠病毒感染人体T细胞作用机制及生物标志物.方法 经过单细胞测序标准化数据整合分析后,将外周血细胞进行聚类注释,选取5种T细胞亚群进行分析.分别计算每个细胞亚群中基因之间的Pearson相关性系数及P值,选取合适的阈值对边进行筛选,并对获得的差异表达基因进行网络映射,构建反映人体T细胞应对新冠病毒感染的基因共表达网络.结果 构建了以真核翻译延伸因子1α1(EEF1A1)基因为中枢的核心共表达网络,网络中的基因表达在新冠病毒感染患者中显著下降,在康复患者中又得以恢复上升.共表达网络和差异分析所得基因的功能富集分析结果表明,新冠病毒感染人体后的T细胞亚群核糖体相关的信号通路和功能本体被显著富集.结论 基于新冠病毒感染患者单细胞测序数据构建的基因共表达网络,发现了 一组以EEF1A1为中枢的具有相似表达模式的功能基因,该基因有望作为新冠病毒感染的诊治标志物.     

急性和慢性髓性白血病细胞中可变剪接事件的识别

目的：为揭示异常的可变剪接在肿瘤中发挥的作用,着重研究急性髓性白血病（ AML）与慢性髓性白血病（CML）内显著差异的可变剪接事件以及剪接异构体。方法基于CML（K562）和AML（THP1、HL－60）细胞系的全转录组测序数据,在全基因组范围内采用TopHat、MATS和Cufflinks计算识别可变剪接事件、重构剪接异构体和分析其差异表达。结果在AML和CML细胞间识别的130个基因在CML细胞系中特异性高表达,80个基因在AML两株细胞系内均表达上调。在CML与AML之间识别了337个差异表达的剪接异构体以及35个差异可变剪接事件。结论 AML与CML细胞系间存在显著差异的可变剪接事件和剪接异构体。白血病相关的可变剪接事件可作为其潜在的标志物或药物靶标。     

肺纤维化相关的细胞间通讯网络研究

目的 建立肺纤维化相关的细胞间通讯网络,并探究细胞间通讯网络在肺纤维化中的作用.方法 基于公开的肺纤维化单细胞测序数据和配体-受体相互作用数据库,并基于自助抽样的新算法建立肺纤维化相关的细胞间相互作用网络;使用Circos和Cytoscape软件对细胞间通讯网络进行可视化,并进一步分析其对细胞功能通路的影响.结果 首先,建立了肺纤维化相关的肌成纤维细胞、成纤维细胞、脂成纤维细胞、间充质祖细胞、间皮细胞、内皮细胞和高表达Pdgfrb的间充质细胞之间的通讯网络模型.然后,筛选出各种细胞类群中与肺纤维化显著相关的配体-受体关系对,并重点研究了几个与肺纤维化有关的配体或受体.最后,分析了与细胞间通讯网络相关的细胞功能通路.结论 该研究基于单细胞转录组测序数据利用自助抽样算法,建立了肺纤维化相关的细胞间通讯网络,并初步揭示了与肺纤维化相关的显著高表达的配体、受体及其影响的功能通路.     

一种基于双重PCR结合毛细管电泳验证转录组测序结果的新方法

目的：将双重PCR与毛细管电泳技术相结合,建立一种验证转录组测序结果的新方法。方法根据前期进行转录组测序的分析结果,挑选8个差异基因作为靶基因,分别使用双重PCR结合琼脂糖凝胶电泳、双重PCR结合毛细管电泳以及实时荧光定量PCR进行验证,比较三者的验证率。结果双重PCR结合琼脂糖凝胶电泳的验证率为50％;双重PCR结合毛细管电泳和实时荧光定量PCR的验证率均为100％。结论双重PCR结合毛细管电泳可作为转录组测序结果验证的一种有效方法。     

某医院船不同舱室微生物多样性初步研究

目的：应用16S rDNA测序技术分析海军某医院船不同舱室细菌群落结构及其多样性,为医院船潜在感染的监测和控制提供科学依据。方法采用拭子涂抹方法在医院船的12个舱室（ A～L位）共采集样本31份,采用IlluminaHiseq2500高通量测序平台PE250测序策略对样品16S rDNA扩增的V3～V4区域进行测序,并进行物种分类、丰度及多样性等生物信息分析。结果31份样品共产生原始测序数据为1860．64 Mbp,每份样品平均产生54．86 Mbp的clean data序列,各样品检测的Tags数量平均为60000,Unique tags序列数量均值为58364;操作分类单元（ operational taxonomic units , OTU）数量181～2239。在医院船12个舱室中共检测到26个细菌门纲目等类,216个细菌科,414个细菌属。其中在各级水平分布的优势菌群分别为厚壁菌门（Firmicutes）、葡萄球菌科（Staphylo－coccaceae ）和葡萄球菌属（ Staphylococcus）。在属水平,葡萄球菌属在L位和J位分布最多,微球菌属在B位分布最多（27．15％）,不动杆菌属在K位占优势（23．83％）,栖水菌属则主要见于C位。12个舱室两间共有的OTU数量占检测到OTU总数的比例均＜22．3％,其中K位有着最高的Chao指数（4711．55）和Shannon－Wiener指数（8．58）,及最高的Simpson优势度指数（0．9905）。结论医院船菌群构成复杂、多样,以革兰阳性葡萄球菌为优势菌属,并有水生环境中的菌属分布,与陆地医院常见细菌分布存在明显差异,而且各舱室细菌物种丰度及分类也存在明显不同。     

Proposal for the standardisation of multi-centre trials in nuclear medicine imaging: prerequisites for a European 123I-FP-CIT SPECT database

Purpose

Multi-centre trials are an important part of proving the efficacy of procedures, drugs and interventions. Imaging components in such trials are becoming increasingly common; however, without sufficient control measures the usefulness of these data can be compromised. This paper describes a framework for performing high-quality multi-centre trials with single photon emission computed tomography (SPECT), using a pan-European initiative to acquire a normal control dopamine transporter brain scan database as an example.

Methods

A framework to produce high-quality and consistent SPECT imaging data was based on three key areas: quality assurance, the imaging protocol and system characterisation. Quality assurance was important to ensure that the quality of the equipment and local techniques was good and consistently high; system characterisation helped understand and where possible match the performance of the systems involved, whereas the imaging protocol was designed to allow a degree of flexibility to best match the characteristics of each imaging device.

Results

A total of 24 cameras on 15 sites from 8 different manufacturers were evaluated for inclusion in our multi-centre initiative. All results matched the required level of specification and each had their performance characterised. Differences in performance were found between different system types and cameras of the same type. Imaging protocols for each site were modified to match their individual characteristics to produce comparable high-quality SPECT images.

Conclusion

A framework has been designed to produce high-quality data for multi-centre SPECT studies. This framework has been successfully applied to a pan-European initiative to acquire a healthy control dopamine transporter image database.     

数字乳腺钼靶X线摄影规范化操作与质量控制

目的 探讨数字化乳腺钼靶X线摄影检查时执行规范化操作与定期质量控制,以提高其图像质量,从而为临床提供高质量影像资料,减少漏诊和误诊.方法 对300例患者(年龄30～75岁)进行乳腺钼靶X线检查,完全按照规范化操作程序并定期质量控制,体位设计选择头尾位(CC)、内外斜位(MLO)或90°侧位(内外侧位ML)所得到的乳腺钼靶数字X线成像(DR)影像进行分析研究.结果 所得数字乳腺钼靶DR图像质量显著提高,乳房组织、腺体结构、腋窝淋巴结及胸大肌等显示清晰,诊断达标率为99％.结论 数字乳腺钼靶X线摄影的规范化操作并定期进行质量控制,可显著提高其图像质量,从而为临床诊断和治疗提供高质量影像资料和依据,提高诊断准确率.     

Use of personal digital assistants for retrieval of medical images and data on high-resolution flat panel displays.

For its new acute care hospital, the University of California at Los Angeles is evaluating innovative technology involving high-resolution flat panel display devices configured as "network appliances" that can be wall mounted for use in the retrieval and display of medical images and data. Physicians and healthcare providers can log on with wireless handheld computers, which can serve as an identification device as well as a navigational tool for selecting patient records and data. These data are displayed and manipulated on the flat panel display without the need for a keyboard or mouse. A prototype was developed with commercially available image display software, which was modified to allow the remote control of software functions from a handheld device through an infrared communication port. The system also allows navigation through the patient data in a World Wide Web-based electronic patient record. This prototype illustrates the evolution of radiologic facilities toward "shareable" high-quality display devices that allow more convenient and cost-effective access to medical images and related data in complex clinical environments, resulting in a paradigm shift in data navigation and accessibility. Copyright RSNA, 2003.     

Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible. However, in order to bring these approaches into forensic laboratories and subsequently as accepted scientific information in a court of law, validated methods will be required to produce and analyze NGS data. We report here on the development of an optimized approach to NGS analysis for the mtDNA genome (mtgenome) using the Illumina MiSeq instrument. This optimized protocol allows for the production of more than 5 gigabases of mtDNA sequence per run, sufficient for detection and reliable reporting of minor heteroplasmic variants down to approximately 0.5–1.0% when multiplexing twelve samples. Depending on sample throughput needs, sequence coverage rates can be set at various levels, but were optimized here for at least 5000 reads. In addition, analysis parameters are provided for a commercially available software package that identify the highest quality sequencing reads and effectively filter out sequencing-based noise. With this method it will be possible to measure the rates of low-level heteroplasmy across the mtgenome, evaluate the transmission of heteroplasmy between the generations of maternal lineages, and assess the drift of variant sequences between different tissue types within an individual.     

In-field whole genome sequencing using the MinION nanopore sequencer to detect the presence of high-prized military targets

ABSTRACT

As DNA sequencing technology advances, in-field genome sequencing capabilities are being explored, utilized and validated. With the assistance of such advances, forensic DNA processing is at the dawn of an era of mobile analyses, as a supplement to the ‘bricks and mortar’ lab model that it is current practice. The prospect of getting a preliminary DNA identification result quickly and in the field can assist in staying on the trail of military targets. This pilot study aimed to assess the capability of the MinION nanopore DNA sequencer in a mobile lab scenario, using as little intervention as possible to evaluate the data produced and the possible impact and interpretation that might arise from it. Results from this study may help guide the direction of further research using this device and relevant software and tools.     

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, DNA testing has historically focused on the recovery of just about 600 base pairs of the mitochondrial DNA control region. Here, we describe our success in recovering complete mitochondrial genome (mtGenome) data (∼16,569 bp) from single shed hairs. By employing massively parallel sequencing (MPS), we demonstrate that particular hair samples yield DNA sufficient in quantity and quality to produce 2–3 kb mtGenome amplicons and that entire mtGenome data can be recovered from hair extracts even without PCR enrichment. Most importantly, we describe a small amplicon multiplex assay comprised of sixty-two primer sets that can be routinely applied to the compromised hair samples typically encountered in forensic casework. In all samples tested here, the MPS data recovered using any one of the three methods were consistent with the control Sanger sequence data developed from high quality known specimens. Given the recently demonstrated value of complete mtGenome data in terms of discrimination power among randomly sampled individuals, the possibility of recovering mtGenome data from the most compromised and limited evidentiary material is likely to vastly increase the utility of mtDNA testing for hair evidence.     

干扰素雾化联合常规治疗对小儿疱疹性咽峡炎的系统评价

目的系统评价干扰素雾化联合常规治疗对小儿疱疹性咽峡炎的疗效。方法计算机检索Cochrane图书馆（2014年第2期）、MEDLLNE、EMBase、Pub Med、中国期刊全文数据库、中国生物医学文献数据库、中文科技期刊全文数据库（各数据库检索时间均从创建至2014年2月）关于干扰素雾化联合常规治疗对小儿疱疹性咽峡炎的随机对照试验。由2名数据员按纳入与排除标准独立筛选试验,并对纳入研究的方法学质量进行评价,提取资料;用Rev Man5.14软件对数据进行Meta分析。结果共纳入14篇随机对照实验,包括2076名患者。结果与对照组比较,干扰素雾化组总有效率优于常规治疗组〔RR=6.04,95%CI（4.36,8.37）,P〈0.00001〕,干扰素雾化组退热时间短于常规治疗组〔MD=-1.30,95%CI（-1.80,-0.80）,P〈0.00001〕,干扰素雾化组疱疹消失时间短于常规治疗组〔MD=-1.77,95%CI（-2.19,-1.36）,P〈0.00001〕,差异均有统计学意义。结论基于现有临床证据,干扰素雾化联合常规治疗对小儿疱疹性咽峡炎有效,安全性好。但由于纳入研究数量较少,研究质量不统一,本结论尚需要更多大样本、高质量临床随机对照试验予以证实。     

Computer-aided administration of a radiology equipment insurance program

The Medical College of Georgia's radiology department has developed computer software that helps the department manage its radiology equipment insurance program. By providing clear menus and choice boxes instead of manual typing wherever possible, the software minimizes the time spent on data entry and management functions. In addition, the software helps track vendor invoices and unreimbursed claims, and maintains a service call log. It also provides a bar-coded service ticket for use by in-house engineers. Completed tickets, which are copied and filed, have replaced ledger books. Furthermore, the software helps the department comply with JCAHO requirements, since service tickets link the documentation of quality control problems to their resolution.     

Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq

Sanger-type sequencing (STS) of mitochondrial DNA (mtDNA), specifically the control region (CR), is routinely employed in forensics in human identification and missing persons scenarios. Yet next-generation sequencing (NGS) has the potential to overcome some of the major limitations of STS processing, permitting reasonable paths forward for full mitochondrial genome (mtGenome) sequencing, while also offering higher-throughput and higher sensitivity capabilities. To establish the accuracy and reproducibility of NGS for the development of mtDNA data, 90 DNA extracts that were previously used to generate forensic quality full mtGenomes using STS were sequenced using Nextera XT library preparation and the Illumina MiSeq. Using the same amplicon product, replicate library sets were generated and sequenced at different laboratories, and analysis was performed in replicate using the CLC Genomics Workbench. Both sequencing sets resulted in 99.998% of positions with greater than 10X coverage when 96 samples (including controls) were multiplexed. Overall, 99.9996% concordance was observed between the NGS data and the STS data for the full mtGenome. The only “discordant” calls involved low level point heteroplasmies, with the differences resulting from stochastic variation and/or the increased sensitivity of NGS. Higher sensitivity also allowed for the detection of a mixed sample previously not detected with STS. Additionally, variant calls were reproducible between sequencing sets and between software analysis versions with the variant frequency only differing by 0.23% and 0.01%, respectively. Further validation studies and specialized software functionality tailored to forensic practice should facilitate the incorporation of NGS processing into standard casework applications. The data herein comprise the largest, and likely most thoroughly examined, complete mtGenome STS-NGS concordance dataset available.     

Quality assessment of randomized controlled trials of contrast media

Numerous randomized controlled trials (RCTs) have been conducted to define the relative benefits of low-osmolality contrast media (LOM) and high-osmolality contrast media (HOM). Because of the clinical and economic significance of the conclusions drawn from these RCTs, the authors used a standardized instrument to evaluate the quality of study design and data analysis of 100 RCTs published between 1982 and 1987 that compared LOM and HOM. The mean quality score (+/- standard deviation) was 39 +/- 12 (maximum possible score, 100). The largest number of patients studied in any RCT was 435; the smallest was five. A majority of the RCTs received high scores on three attributes of quality, intermediate scores on seven, and low scores on nine. These results underscore the difficulty of designing, performing, analyzing, and reporting high-quality RCTs. Nevertheless, limitations in study design and data analysis need to be considered when interpreting results of these RCTs. Future RCTs comparing LOM and HOM should be performed with greater attention to basic elements of good study design and data analysis.