新一代高通量测序技术及其法医学应用前景

随着大规模基因组学的兴起,多种高通量、低成本的新一代测序技术应运而生,并逐步被应用到生命科学研究的各个领域.本文对新一代测序技术进行综述介绍,并对其在法医学领域中的应用前景进行展望.     

运用二代测序技术拆分混合STR分型

目的探讨二代测序技术在混合STR分型拆分中的应用。方法对一例强制猥亵妇女案中受害人颈部的拭子和血样作DNA提取,以Precision ID GlobalFiler^TM NGS STR Panel v2试剂盒制备文库,经Ion S5测序仪测序,运用Torrent_Suite_v5.2.1软件进行数据分析后,将检测基因座的序列多态STR分型与长度多态STR分型进行比较。结果在D8S1179、D21S11、D2S441、D2S1338、D10S1248五个基因座发现存在序列特异的等位基因亚型,利用这些亚型对混合STR分型进行了成功拆分。结论二代测序技术提供的等位基因序列信息可对混合STR分型的拆分起到帮助作用。     

A rape case solved by mitochondrial DNA mixture analysis

In cases of stains that contain mixed DNA from different contributors, analyzing mitochondrial DNA (mtDNA) requires the use of cloning techniques. We developed an efficient cloning technique that was applied in a rape case. After a differential lysis-based DNA extraction from vaginal swabs, hypervariable region I and II (HVI, HVII) amplicons obtained from the male fraction were cloned. Although we mainly found the victim's haplotype, we were able to detect the suspect's haplotype in two clones for HVI and in one clone for HVII. As the midpiece of the flagellum, which contains mitochondria, can be lost during the differential lysis, we also investigated the female fraction by cloning to evaluate the proportion of victim/suspect mtDNA. Unfortunately, only clones presenting the victim's haplotype were found. This case highlights the need for an optimal differential lysis protocol to enrich the male fraction not only with nuclear but also mitochondrial DNA.     

二代测序技术在Y染色体遗传标记分型中的法医学应用

法医遗传学领域常利用Y染色体的父系遗传特点,对非重组区遗传标记进行检测并用于亲缘关系鉴定、混合斑检测、家系排查以及种族推断等研究.目前毛细管电泳仍是应用最为广泛的检测技术,基于该技术的商业化检测试剂盒及数据分析处理系统十分成熟.随着生物信息量的增长,传统检测技术通量低的弊端逐渐显现,推动了法医DNA分型技术的革新.近年...     

二代测序技术在法医学中的应用

随着二代测序技术的快速发展,其高通量和低成本在生命科学领域应用广泛,测序的通量更高,测序时间和成本不断下降,使得其被广泛应用于微生物研究、古DNA研究、临床诊断、法医学研究等。本文阐述了二代测序技术平台及其遗传标记在法医学中的应用,包括STR分型、SNP分型、HLA基因型预测以及在降解检材中的应用等。     

基于下一代测序的全解析度STR分型研究进展与展望

下一代测序技术具有高通量、高速度、集成化、低成本等显著优势,近年来已在科研和临床诊断领域得到广泛应用,在法医遗传学领域亦具有重要应用前景。当前主流的STR分型方法仅关注序列的长度多态性,然而由于核心重复结构存在差异或扩增区段内存在SNP,序列长度相等的等位基因可能是具有遗传稳定性的完全不同的等位基因,此类STR序列多态性是个体识别或亲缘关系分析的宝贵资源。基于下一代测序的STR分型在现有数据输出方式基础上,允许进一步关注STR的序列多态性,对STR基因座进行全解析度分型,显著提升STR基因座的个体识别能力。本文以法医STR遗传标记和下一代测序技术为关注焦点,系统综述基于下一代测序的全解析度STR分型领域国际最新研究进展,深入探讨该技术在法医DNA实验室的实际应用潜力和可能面临的挑战,希冀对相关研究和实践提供参考。     

24个Y-STR基因座在云南苗族人群中的序列多态性

目的研究法医学常用Y-STR基因座在中国云南苗族男性个体中的序列多态性。方法采用M48磁珠提取纯化试剂盒提取样本DNA,使用ForenSeqTM DNA Signature Prep试剂盒制备文库,Miseq FGx平台进行测序,ForenSeq Universal Analysis v1.2.1软件进行数据分析,用Arlequin v3.5软件计算各Y-STR基因座相关的统计学参数,将Y-STR基因座长度多态性与序列多态性进行比较。结果108名云南苗族个体中共检出106种单倍型,总体单倍型多样性(HD)和Y-STR分型系统的分辨能力(DC)分别为0.9993和0.9815。24个Y-STR基因座共检出204个基因,基因多样性(gene diversity,GD)值为0.2177~0.9481,15个Y-STR基因座的GD值大于0.6。DYF387S1、DYS390、DYS389II、DYS437、DYS438、DYS448、DYS612基因座存在长度相同的基因核心序列不同的情况。结论该24个Y-STR基因座在云南苗族男性人群中具有丰富的遗传多态性。研究结果可为Y-STR数据库的建立、群体遗传学和法医学实践提供参考。     

The Recovery and Analysis of Mitochondrial DNA from Exploded Pipe Bombs*

Abstract: Improvised explosive devices (IEDs) represent one of the most common modes of arbitrarily injuring or killing human beings. Because of the heat generated by, and destruction to, an IED postconflagration, most methods for identifying who assembled the device are ineffective. In the research presented, steel pipe bombs were mock‐assembled by volunteers, and the bombs detonated under controlled conditions. The resultant shrapnel was collected and swabbed for residual cellular material. Mitochondrial DNA profiles were generated and compared blind to the pool of individuals who assembled the bombs. Assemblers were correctly identified 50% of the time, while another 19% could be placed into a group of three individuals with shared haplotypes. Only one bomb was assigned incorrectly. In some instances a contaminating profile (mixture) was also observed. Taken together, the results speak to the extreme sensitivity the methods have for identifying those who assemble IEDs, along with precautions needed when collecting and processing such evidence.     

Routine forensic use of the mitochondrial 12S ribosomal RNA gene for species identification

Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.     

法医学二代测序STR分型准确度与测序深度的关联性评估

目的利用实验数据对法医学二代测序STR分型测序深度与分型结果准确度的关联性进行评估。方法使用商业化基因组DNA制备单一来源和混合的DNA样本,以Thermo Fisher公司的25重早期测试试剂盒进行目的STR片段扩增,每种扩增产物分别使用4种不同的序列标签平行建库,并控制标记每一种序列标签的文库上机量依次占一张Ion 318芯片的1/4、1/8、1/16、1/32。经Ion PGMTM基因测序仪测序,以及Ion Torrent SuiteTM软件进行数据分析;同时对庞敬博等人发表的基于相同试剂盒和测序仪检测的95名中国汉族无关个体的6928条等位基因、影子峰和噪音序列进行测序深度统计分析,寻找测序深度与STR分型准确度的关联性。结果各基因座测序深度随文库上样量减少而呈明显下降趋势。对于单一来源样本,每张芯片上样不超过8个均一化文库可实现全部基因座的完整分型;对于1∶20比例的混合DNA,每张芯片上样不超过4个均一化文库时,未发现微量组分的等位基因丢失。人群数据测序深度统计显示,该体系基因座间存在不均衡性,有必要针对各基因座分别设定分析阈值参数。结论测序深度与法医学STR分型结果的准确性密切相关,各基因座最低测序深度与平均测序深度的比值可作为设定分析阈值的重要参考指标。本研究确定的单张芯片上样数量仅适用于本实验体系,但相关实验设计和方案可供其他实验体系开展类似工作参考。     

The Conviction of Dr. Crippen: New Forensic Findings in a Century‐Old Murder

Abstract: Dr. Hawley Crippen was accused and convicted of murdering his wife in London in 1910. Key to the conviction was microscopic analysis of remains found in the Crippen’s coal cellar, which were identified as Cora Crippen based on a scar she was said to have. Dr. Crippen was hanged, always proclaiming his innocence. In this study, genealogical research was used to locate maternal relatives of Cora Crippen, and their mitochondrial haplotypes were determined. Next, one of the pathology slides of the scar was obtained, DNA was isolated, and the haplotype was determined. That process was then repeated. Finally, both DNA isolates were assayed for repetitive elements on autosomes and repetitive elements specific to the Y chromosome. Based on the genealogical and mitochondrial DNA research, the tissue on the pathology slide used to convict Dr. Crippen was not that of Cora Crippen. Moreover, that tissue was male in origin.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.