基于Ion Torrent PGM~(TM)测序系统的人mtDNA全测序分析

目的应用Ion Torrent PGM~(TM)测序系统对人线粒体DNA(mitochondria DNA,mtDNA)全序列进行分析检测,研究不同组织间mt DNA序列差异情况。方法通过法医尸体检验采集6名无关个体的组织样本,包括胸腔血液、头发、肋软骨、指甲、骨骼肌和口腔上皮。使用4对引物对线粒体全序列进行扩增,应用Ion Shear~(TM)Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM~(TM)测序系统上进行线粒体基因组全序列测序,并针对异质性位点和在HVⅠ区域突变位点,进行Sanger测序验证。结果所有样本的全基因组mtDNA都扩增成功,6名无关个体分属于6种不同的单倍型,同一个体不同组织之间mtDNA存在异质性差异。异质性位点和HVⅠ区域突变位点采用Sanger测序结果均得到验证。通过Kappa统计方法进行一致性检验后发现,相同个体不同组织的mtDNA序列检验结果仍具有较好的一致性。结论本研究所采用的人线粒体基因组全序列的测序检验方法,可以检测出同一个体不同组织间mtDNA的异质性差异,该差异具有较高的一致性,该结果对mtDNA在法庭科学中的应用具有指导作用。     

口蹄疫病毒A/WH/09株结构蛋白P1基因的克隆及其B细胞抗原表位的预测

对口蹄疫病毒(FMDV)A/WH/09株P1结构蛋白基因进行了扩增、克隆及序列测定。结果显示,FMDV A/WH/09株P1基因长2 208bp,包含完整的开放阅读框,编码736个氨基酸,其中VP1长636bp,编码212个氨基酸;VP2长654bp,编码218个氨基酸;VP3长663bp,编码221个氨基酸;VP4长255bp,编码85个氨基酸。采用DNAStar Protean软件对P1蛋白的二级结构、可塑性、亲水性、表面可及性及抗原指数等参数进行了综合分析,并预测其B细胞的抗原表位分布。结果表明,VP1、VP2和VP3上均有多个区域为B细胞抗原优势表位,VP4上也有少量潜在的B细胞抗原表位。与已鉴定的B细胞抗原表位相比,本试验所用方法预测的结果有较高的准确度,是一种较为先进的表位研究方法。     

Failed agreements and their impact on subsequent mediation onset and success in intrastate conflicts

ABSTRACT

What impact do failed mediated agreements have on subsequent mediation onset and success? The question of mediation onset and success is undoubtedly important, given that mediation is one of the international community’s preferred conflict management tools, but its voluntary nature leaves room for the conflict parties to (dis)agree to talks and possible settlements. Existing research suggests that previous mediation outcomes can affect subsequent mediation efforts positively or negatively – depending on the outcome in focus. This article argues that failed agreements – an outcome of mediation that has not been accounted for in existing literature – underscore the persistence of the commitment problem, and therefore the hazards of sharing private information. Consequently, the conflict parties question the utility of mediation, and the likelihood of subsequent mediation onset decreases. If subsequent talks take place despite the failed agreement, the conflict parties refrain from sharing private information, and reaching an agreement becomes thus less likely. Drawing on the Uppsala Conflict Data Program’s (UCDP) Managing Intrastate Conflict in Africa data set and the UCDP Peace Agreement data set, the results strongly underline the negative impact failed agreements have on subsequent mediation onset, and thereby show that agreement longevity is crucial not only for peace duration but also for leaving the door open to subsequent talks.     

Exploitation of the Ultraviolet Properties and Machine Cut Edges of Paper to Associate and Sequence Sheets in a Ream

Previously unreported line patterns visible under ultraviolet light were observed on a proportion of plain white A4 printer/copier paper from different manufacturers. These Ultraviolet Line Patterns (UVLPs) usually appear as stripes down the vertical length of the paper. Typically, the UVLPs were found to “repeat” through the ream in a predictable way, while also changing. It is postulated that the repeating nature of the UVLPs is a result of the way that paper is manufactured. This leads to the ability to sequence the sheets compared to their original source paper. Even in the absence of UVLPs, it is possible to use our observation of the manufacturing process to anticipate the order of several sheets of paper and conclusively associate them, in some cases, by physically fitting their machine cut edges and crossing paper fibers. Such a novel approach to examining questioned documents would be highly useful in forensic casework.     

基于二代测序的混合检材精细化分型研究

目的基于二代测序平台进行混合检材精细化STR分型,并评估其法医学应用价值。方法收集性侵案件中3例混合检材及其比对样本,采用M48磁珠提取纯化试剂盒提取样本DNA,使用Foren SeqTM DNA Signature Prep试剂盒制备文库,Mi Seq FGx平台进行测序,Foren SeqTM Universal Analysis v1.2.1软件进行数据分析,将STR序列多态分型与长度多态分型进行比较。结果对3例混合检材STR分型进行拆分,在D3S1358、D13S317与D9S1122基因座发现存在同一长度多态等位基因包含两个个体的序列多态等位基因的情况。结论二代测序技术可对混合检材进行精细鉴别,为混合分型数据拆分提供更多线索和依据。     

Soil sample metagenome NGS data management for forensic investigation

Soil analysis is a valuable resource in forensic investigation. Classical forensic soil analysis involves examination of its physical characteristics and chemical composition, such as soil type, colour, particle size, shape, pH, elemental, mineral and organic content. However the limited variability of these parameters is not always allowing adequate discrimination between soil samples. As soil supports extreme diversity of microorganisms and eukaryotic communities, microbiological approaches have been proposed. Several molecular approaches for microbial DNA profiling are available; however there is a lack of published data of implementation of the next generation sequencing (NGS) approaches for forensic soil analysis.The aim of the current study was elaboration of criteria for soil metagenome data management and database searching. We used our previously sequenced collection of 11 samples collected from different environments (forests, fields, grasslands, urban park) with different flora. The single sample collection includes 9 soil samples per one sampling area (30 m × 30 m) spaced by 15 m. In the current study we concentrated mainly on 18S rRNA gene V2-V3 region for fungi however SSU rRNA region for arbuscular mycorrhizal (AMF) fungi and V2-V3 hypervariable region of 16S rRNA gene for bacterial communities were taken into account. The sequencing was performed by Roche/454 platform. For data analysis OTU based approach on mothur software and NCBI BLASTN search were used. NCBI BLASTN analysis revealed altogether 2983 AMF matches and 8997 18S matches as well as 25477 OTUs (16S) were determined. Several data filtration approaches were used for data management. We found that 18S marker results could be used to create and run a filtered database that is computationally much more efficient and flexible. Our results have broad impact; however more samples have to be analysed, additional studies performed and cooperation between soil scientists and forensic scientists is required to be able to implement these novel techniques into the routine forensic practice.     

马动脉炎病毒大囊膜蛋白和膜蛋白基因的克隆与序列分析

参考GenBank收录的马动脉炎病毒(EAV)读码框大囊膜蛋白基因 ORF5、膜蛋白基因ORF6的核苷酸序列,分别设计了2对引物,对EAV的这两种主要结构蛋白基因进行了 RT PCR;将扩增产物克隆于pUC18通用载体,对重组质粒进行限制性内切酶分析和基因测序,证实克隆片段的可靠和准确性。测序结果,ORF5目的片段包含768 bp,编码255个氨基酸组成的多肽;ORF6目的片段包含489 bp,编码162个氨基酸组成的多肽。与EAV NC 002532标准毒株比较,ORF5、ORF6核苷酸的同源性分别为99.1%和99.4%;推导氨基酸的同源性分别为96.9%和98.2%。     

Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses

DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa “Brazuka” and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa “Brazuka” with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.