下一代测序技术在感染性疾病中的应用进展

感染性疾病是目前全世界患者人群中发病率和死亡率居高不下的重要原因之一.传统的实验室检测方法存在较多局限性,如检测时间长、培养阳性率低等.而下一代测序技术是一种无偏倚检测方法,其最新进展以及准确全面的数据库创建,使得NGS技术的应用跨越了从微生物研究到诊断微生物学的界限.     

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。     

CT影像质量评价和质量控制

众所周知 ,ＣＴ检查已在临床诊断治疗中成为不可缺少的检查方法 ,ＣＴ诊断主要是依据ＣＴ影像所提供的正常或异常的信息而作出诊断结论 ,可见优质ＣＴ影像是诊断确切性的前提条件 ,因此对ＣＴ影像质量的评价就成为十分关注和探讨的问题[1 3 ] 。一、ＣＴ影像 (片 )的优劣评价应考虑以下几个方面1.满足ＣＴ诊断要求的影像信息。例如 ,颅脑扫描的脑灰质和脑白质的区分 ;肝脏扫描的肝血管界线分明 ;腰椎椎间盘扫描的椎间盘和两侧神经根的显示。2 .适当的影像密度定量。包括背景密度 [组织影像外的最大密度 (Ｄｍａｘ)和最小密度 (Ｄｍｉｎ) ]和…     

乳品质量的微观控制

目前,国外出现了利用微观结构控制乳品质量的新动向。研究发现:乳制品的微观结构决定产品的粘性、弹性、胶体性、硬度、细致度、口感和外观等物理特性,而这些特性影响着消费者的购买心理和市场潜力。 乳品的微观结构主要取决于酪蛋白胶粒和脂肪球这两种乳基本微粒物质。在电镜下酪蛋白胶粒直径平均为100μm,在高倍光镜或电镜下脂肪球直径平均为2.58μm。不同的加工处理因素,会造成两者产生不同的大小,排列结构变化等,从而表现出不同的质量特征。将微结构变化与加工因素和质量特征联系起来,就可以找出加工条件对产品物理特性的影响关系,达到控制产品质量的目的。     

血清PSA测定质量控制分析研究

如何保证自动化免疫分析测定质量 ,使测定结果有良好的重复性及可比性 ,对科研及临床应用都具有重要意义。本研究结合参加ＩＡＥＡ的ＰＳＡ测定外部质量评价 (ＥＱＡ)课题 (ＩＡＥＡＲｅｓｅａｒｃｈＣｏｎｔｒａｃｔＮｏ 93 17/ＲＩ) ,对 1998年 5～ 12月间常规测定中内、外质量评估参数进行了分析 ,并对测定质量中出现的问题提出了改进意见。材料与方法1 仪器。ＥＧ＆ＧＷａｌｌａｃ自动时间分辨荧光免疫分析仪 (ＡｕｔｏＤＥＬＦＩＡ 12 3 5 )。2 试剂。总ＰＳＡ (Ｔ ＰＳＡ)及游离ＰＳＡ(Ｆ ＰＳＡ)测定采用Ｗａｌｌａｃ公司Ｐｒｏｓｔ…     

AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data

The feasibility of generating mitochondrial DNA (mtDNA) data has expanded considerably with the advent of next-generation sequencing (NGS), specifically in the generation of entire mtDNA genome (mitogenome) sequences. However, the analysis of these data has emerged as the greatest challenge to implementation in forensics. To address this need, a custom toolkit for use in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed through a collaborative effort between the Armed Forces Medical Examiner System − Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics. The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and includes the interpretation range required for mtDNA data reporting. AQME also integrates an mtDNA haplogroup estimate into the analysis workflow, which provides the analyst with phylogenetic nomenclature guidance and a profile quality check without the use of an external tool. Supplemental AQME outputs such as nucleotide-per-position metrics, configurable export files, and an audit trail are produced to assist the analyst during review. AQME is applied to standard CLC outputs and thus can be incorporated into any mtDNA bioinformatics pipeline within CLC regardless of sample type, library preparation or NGS platform.An evaluation of AQME was performed to demonstrate its functionality and reliability for the analysis of mitogenome NGS data. The study analyzed Illumina mitogenome data from 21 samples (including associated controls) of varying quality and sample preparations with the AQME toolkit. A total of 211 tool edits were automatically applied to 130 of the 698 total variants reported in an effort to adhere to forensic nomenclature. Although additional manual edits were required for three samples, supplemental tools such as mtDNA haplogroup estimation assisted in identifying and guiding these necessary modifications to the AQME-generated profile. Along with profile generation, AQME reported accurate haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup assignment, although phylogenetically close, identified a bug that only affects partial mitogenome data. Future adjustments to AQME’s haplogrouping tool will address this bug as well as enhance the overall scoring strategy to better refine and automate haplogroup assignments. As NGS enables broader use of the mtDNA locus in forensics, the availability of AQME and other forensic-focused mtDNA analysis tools will ease the transition and further support mitogenome analysis within routine casework. Toward this end, the AFMES-AFDIL has utilized the AQME toolbox in conjunction with the CLC Genomics Workbench to successfully validate and implement two NGS mitogenome methods.     

粉刺净胶囊剂质量控制研究

采用薄层色谱法对制剂中的大黄、赤芍进行了定性鉴别 ;用消化—配位滴定法对硫酸锌进行了含量测定 ,方法简便 ,专属性及重现性好 ,可作为该制剂的质量控制指标。     

SEQ Mapper: A DNA sequence searching tool for massively parallel sequencing data

The development of massively parallel sequencing (MPS) has increased greatly the scale of DNA sequencing. The analysis of massive data-files from single MPS analysis can be a major challenge if examining the data for potential polymorphic loci. To aid in the analysis of both short tandem repeat (STR) and single nucleotide polymorphisms (SNP), we have designed a new program called SEQ Mapper to search for genetic polymorphisms within a large number of reads generated by MPS. This new program has been designed to perform sequence mapping between reference data and generated reads. As a proof-of-concept, sequences derived from the allelic ladders of five STR loci and data from the amelogenin locus were used as reference data sets. Detecting and recording the polymorphic nature of each STR loci was performed using four levels of search criteria: the entire STR locus spanning the two primers; the STR region plus the two primer sequences; the STR region only; and the two primers only. All the genotypes of 5 STR loci and the amelogenin gene were identified correctly using SEQ Mapper when compared to results obtained from capillary electrophoresis based on 10 test samples in this study. SEQ Mapper is a useful tool to detect STR or SNP alleles generated by MPS in both clinical medicine and forensic genetics.     

FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise

Massively parallel sequencing (MPS) is on the advent of a broad scale application in forensic research and casework. The improved capabilities to analyse evidentiary traces representing unbalanced mixtures is often mentioned as one of the major advantages of this technique. However, most of the available software packages that analyse forensic short tandem repeat (STR) sequencing data are not well suited for high throughput analysis of such mixed traces. The largest challenge is the presence of stutter artefacts in STR amplifications, which are not readily discerned from minor contributions. FDSTools is an open-source software solution developed for this purpose. The level of stutter formation is influenced by various aspects of the sequence, such as the length of the longest uninterrupted stretch occurring in an STR. When MPS is used, STRs are evaluated as sequence variants that each have particular stutter characteristics which can be precisely determined. FDSTools uses a database of reference samples to determine stutter and other systemic PCR or sequencing artefacts for each individual allele. In addition, stutter models are created for each repeating element in order to predict stutter artefacts for alleles that are not included in the reference set. This information is subsequently used to recognise and compensate for the noise in a sequence profile. The result is a better representation of the true composition of a sample. Using Promega Powerseq™ Auto System data from 450 reference samples and 31 two-person mixtures, we show that the FDSTools correction module decreases stutter ratios above 20% to below 3%. Consequently, much lower levels of contributions in the mixed traces are detected. FDSTools contains modules to visualise the data in an interactive format allowing users to filter data with their own preferred thresholds.     

Examination of the usefulness of next-generation sequencing in mixed DNA samples

The identification of individuals from mixed DNA samples is an important application of DNA typing. Although the discriminatory power of DNA profiling has improved dramatically, a limiting factor is that individuals cannot be identified via short tandem repeat (STR) analysis. We used next-generation sequencing (NGS) to examine the mixed DNA samples. Our results showed that STR nucleotide sequences and single nucleotide polymorphisms (SNPs) analysis via NGS may enable the identification of each distinct subject from a DNA mixture containing DNA of the victim and suspect.     

Short tandem repeat typing on the 454 platform: Strategies and considerations for targeted sequencing of common forensic markers

To investigate the feasibility of next generation sequencing technology (NGS) for the multiplex detection and sequence production of short tandem repeats (STRs) from degraded and low DNA quantity samples, standard polymerase chain reaction amplification methods were used to enrich for commonly employed STR markers. Samples were amplified with two multiplexing strategies: a multiplex containing thirteen miniSTR markers and a series of multiplexes containing four miniSTR markers each. Each sample multiplex was barcoded with a sample-specific multiplex identifier for subsequent parallel tagged sequencing on the GS Junior System (454 Life Sciences, a Roche company, Branford, CT). Sequencing results from over fifty DNA extracts representing both pristine samples and low-quality evidentiary specimens reflected known genotypes and were consistent across multiple extracts and/or amplifications of the same sample. Furthermore, the NGS data revealed sequence information not available with standard capillary electrophoresis-based detection alone. For the population samples tested, a total of 152 single nucleotide polymorphisms or insertions/deletions were identified in over 935 recovered alleles, averaging one polymorphism for every six recovered alleles. For three of the loci, the sequence information doubled the number of alleles detected via traditional STR typing by fragment analysis. In addition, twenty-eight of these variants were only seen once within our dataset, highlighting the potential for discrimination among individuals. These additional data are likely to be particularly valuable in missing persons and disaster victim identification cases for which only partial profiles may be recovered and/or only distant kin are available as references. And, considering the opportunity to target only small amplicons with NGS, this type of STR typing will allow for greater information recovery from challenging casework samples. While our results highlight the potential of new technologies for recovering discriminatory genetic information from evidentiary specimens, our data also reveal the complexities of NGS-based STR typing, both in terms of the laboratory assays themselves as well as the downstream data processing and analysis.     

A global snapshot of current opinions of next-generation sequencing technologies usage in forensics

The future of forensic DNA testing is being shaped by the research and usage of next-generation systems, which have increased the multiplexing capabilities of the field and the type and amount of genetic data that can be utilized for investigations. The NGS adoption for casework has been slow, albeit the plethora of data that has been published. This study evaluated the current opinions on sequencing in forensics. A 20-question online-survey focusing on NGS knowledge, training, and usage was distributed to 6001 forensic DNA researchers and practitioners worldwide. A total of 367 responses were obtained from all continents (North/South America (69.8%), Europe (21.2%), Asia (5.5%), Oceania (2.5%), and Africa (1%)). The respondents consisted of 50% practitioners, 31% researchers, and 19% both. Of these, 38% already own a next-gen sequencing instrument, and 13% are planning to purchase one. Overall, there exists an extensive knowledge on next-gen sequencing within the forensic community, including among laboratories that have not yet implemented this high-throughput technology in their workflows. Current usage focuses primarily on SNP analysis for investigative leads and mitochondrial DNA analysis while future applications included both STR and SNP testing applied to general casework. The major overall concerns respondents have for implementing a sequencing instrument include limited funding, staffing, lack of time, and the cost-effectiveness of providing this service. Specific technical concerns that the respondents had are the lack of training, statistical applications, bioinformatics support, and of rigorous guidelines and recommendations. Most of the respondents do believe there will be a technology shift from using CE only to the use of NGS on casework in 5–10 years. In addition, around 66% of respondents believe that it is moderately to very likely that the court will accept sequencing analysis. Sixteen percent fell in the middle, and the remaining 15% believe it is more unlikely, with 3% of respondents believing it is very unlikely. In conclusion, this work outlines current analytical challenges experienced by the global forensic DNA community and addresses different strategies for the implementation of next-gen sequencing technologies in casework.     

宏基因二代测序在中枢神经系统感染性疾病诊断中的应用

 目的 探讨宏基因二代测序(metagenomic next-generation sequencing,mNGS)在中枢神经系统感染性疾病诊断中的应用价值。方法 回顾分析2019-01至2021-12山西白求恩医院神经内科收治的85例中枢神经系统感染性疾病患者,系统收集各项资料,探究mNGS在该类疾病诊断应用中的优势。结果 85例中,mNGS检出病原体21种,检出阳性57例,其中与最终诊断符合48例,不符合9例,准确率为56.5%(48/85);传统方法检出阳性23例,其中与最终诊断符合22例,不符合1例,准确率25.9%(22/85),两种方法准确率差异有统计学意义(P＜0.05)。结论 较传统方法而言,宏基因二代测序对中枢神经系统感染性疾病的准确率高,可以作为传统方法的互补方法,但因mNGS检出结果与最终诊断不符情况较多,检测结果的判读应结合临床慎重考虑。     

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.