Current Next Generation Sequencing technology may not meet forensic standards

In a Nature paper of 2010, the concern was raised that intra-individual mtDNA variation may be more pronounced than previously believed, in that heteroplasmies are common and vary markedly from tissue to tissue. This claim taken at face value would have considerable impact on forensic casework. It turns out however that the employed technology detected the germ-line variation relative to the reference sequence only incompletely: on average at least five mutations were missed per sample, as an in silico reassessment of the data reveals. Before one can really set out to access to entire mtDNA genome data with relative ease for forensic purposes, one needs careful calibration studies under strict forensic conditions—or might have to wait for another generation.     

Single primer extension (SPEX) amplification to accurately genotype highly damaged DNA templates

High levels of non-authentic sequence data can be generated by traditional PCR-based methodologies when DNA is damaged, template numbers are small and/or the target amplification size too large. We therefore present an alternate methodology based on single primer extension (SPEX) amplification; that places no pre-defined size constraints on amplification and interacts with only one of the DNA strands at the target locus.     

GAMarker基因分型专家系统的设计与实现

目的DNA基因分型软件是DNA检测技术体系不可缺少的一环,为拓展GA118系列遗传分析仪器的应用,须研制一套DNA基因分型专家系统,以满足法庭科学DNA检验鉴定工作的需要。方法基于已掌握的DNA片段定长和基因分型数据处理解决方法及相关核心算法,使用JAVA语言和MYSQL数据库,利用Maven进行项目管理,经对GA118系列、ABI系列数据文件解析和数据分析,研发了专家系统GAMarker。结果该系统实现了样本和数据呈现、样本分析要素质量评估、分析方法管理、分型结果展示和人工核查、电泳数据审查、生成分析报告、系统安全等功能,并可分析8色荧光数据。结论GAMarker可进行软件设定、数据分析与比对、图谱查看与编辑,是一套完整的DNA片段分析流程和直观的数据审核工具,可代替国外产品、有效支撑国产遗传分析仪相关系列型号的数据分析,能满足侦破案件、DNA数据库建设的需要。     

车辆上脱落细胞STR检验

目的对汽车方向盘及变速杆把手上的皮肤脱落细胞进行STR检验研究。方法用EZ-tape采集车辆方向盘及变速杆上的脱落细胞,Chelex-100法与磁珠法结合提取DNA,延长保温时间。定量后调整PCR反应体系,适当增加PCR循环数,增加PCR产物量及延长进样时间进行电泳检测,使用GeneMapperIDV3.2软件进行STR分型。同时,对比使用多重置换扩增技术对提取的DNA进行全基因组扩增。结果对33份车辆方向盘和变速杆上脱落细胞的检测,其中19份检出全部基因座的基因分型结果,9份检出部分基因型,5份未检出DNA分型结果。而利用多重置换扩增技术未获得满意图谱。结论本研究建立的脱落细胞收集、DNA提取、PCR扩增及检测方法适合于车辆上脱落细胞的检验,其结果优于使用多重置换扩增技术获得的分型。     

车辆上脱落细胞STR检验

目的对汽车方向盘及变速杆把手上的皮肤脱落细胞进行STR检验研究。方法用EZ-tape采集车辆方向盘及变速杆上的脱落细胞,Chelex-100法与磁珠法结合提取DNA,延长保温时间。定量后调整PCR反应体系,适当增加PCR循环数,增加PCR产物量及延长进样时间进行电泳检测,使用GeneMapperIDV3.2软件进行STR分型。同时,对比使用多重置换扩增技术对提取的DNA进行全基因组扩增。结果对33份车辆方向盘和变速杆上脱落细胞的检测,其中19份检出全部基因座的基因分型结果,9份检出部分基因型,5份未检出DNA分型结果。而利用多重置换扩增技术未获得满意图谱。结论本研究建立的脱落细胞收集、DNA提取、PCR扩增及检测方法适合于车辆上脱落细胞的检验,其结果优于使用多重置换扩增技术获得的分型。     

Statistical analysis tools of mixture DNA samples: When the same software provides different results

The high complexity of the genetic analysis of crime scene samples is mainly related to the unknown number of contributors, low DNA quantity and quality, and associated stochastic effects. The difficulty and subjectivity of interpreting casework samples was the motto for the development of software to mitigate these conditions and allow the quantification of the genetic evidence. Currently, there are several tools for statistical analysis of mixture samples based on either qualitative or quantitative models. The first considers the electropherograms’ qualitative information, while the latter also considers the associated quantitative information. This work’s main goal was to evaluate the effect that parameters’ settings variation may have on the LR computation, specifically the drop-in frequency parameter. For that, a qualitative – LRmix Studio – and two quantitative software – STRmix™ and EuroForMix – were considered and an intra-software analysis was performed, using as input real casework samples. The drop-in frequency variation showed an impact, leading to differences higher than four units (log₁₀ scale) for some pairs of samples. In addition, for some cases, no comparisons were performed either because the tool computed a null LR value or displayed an error message. Thus, this work reinforces the importance of proper parameters’ modeling and estimation in forensic casework evaluation.     

Simultaneous detection of ABO and Secretor-nonsecretor blood groups from forensic biological samples by fragment analysis

This paper describes the simultaneous detection of ABO and Secretor-nonsecretor (SE) blood groups from forensic biological samples by fragment analysis using the ABI PRISM^® 3130 genetic analyzer. The method allows the assay of well-known base changes at three nucleotide positions 261, 796 and 803 on cDNA of the ABO gene, and at 385 and 428 on cDNA of SE gene and a SE pseudo gene, so that reliable group prediction is established by the presence of representative alleles. As a result, simultaneous detection of ABO and SE blood groupings from biological samples was correctly determined by our methods.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR^® Identifiler^® amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR^® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

A production system to generate reference genetic profiles from buccal swab cells on FTA cards

To generate the large quantity of genetic profiles that continuously feeds the French Reference Sample Database (F.N.A.E.G.), a secure and robust process is required. A complete automated production chain has been developed by Hamilton Robotics and the French Police Scientifique in Lyon to produce genetic profiles from buccal swab cells on FTA^® cards. The activities have been divided between Pre- and Post-PCR. The samples on FTA^® cards are punched directly into PCR plates. The plates are then transferred onto the Hamilton Microlab^® STAR liquid handling instruments. Air displacement pipetting coupled with Hamilton's CO-RE Technology allows a highly secure and contamination free pipetting for FTA^® wash and STR^® Identifiler^® PCR reaction set-up. Plates are sealed and transferred to the Post-PCR system for pooling into 384 format and denaturation. Throughout the process steps a complete sample tracking is performed and sample information is transferred to the LIMS system. This poster describes in detail this production system with an actual throughput of 40,000 samples/month, which is in production since 2006.