Analyzing degraded DNA and challenging samples using the ForenSeq™ DNA Signature Prep kit

Typing short tandem repeats (STRs) is the basis for human identification in current forensic testing. The standard method uses capillary electrophoresis (CE) to separate amplicons by length and fluorescent labeling. In recent years new methods, including massively parallel sequencing (MPS), have been developed which increased the discriminative power of STRs through sequencing. MPS also offers the opportunity to test more genetic markers in a run than is possible with standard CE technology. Verogen’s ForenSeq™ DNA Signature Prep kit includes over 150 genetic markers [STRs and single nucleotide polymorphisms (SNPs)]. Further, MPS separation depends on sequences rather than lengths; therefore, amplicons can be small or even of the same lengths. These improvements are advantageous when testing challenging forensic samples that could be severely degraded.This study tested the ForenSeq™ DNA Signature Prep kit in repeated experimental runs on series of degraded DNA samples, ranging from mild to severe degradation, as well as 24 mock case-type samples, derived from bones, blood cards, and teeth. Despite passing the quality metrics, positive controls (2800 M) showed drop-outs at some loci, mostly SNPs. Sequencing DNA samples repeatedly in two experimental runs as well as sequencing one pooled library in triplicate led to the assumption that spurious alleles of the Y-STRs in this study were not a result of sequencing artifacts but could be due to sequence structures (e.g. duplications, palindromes) of the Y-chromosome and/or might be accumulated during library preparation.Two sets of serially degraded DNA samples revealed that dropped-out loci were primarily loci with long amplicons as well as low read numbers (coverage), e.g. PentaE, DXS8378, and rs1736442. STRs started to drop out at degradation indices (DIs) > 4. However, severely degraded DNA (DI: 44) still resulted in 90% of the 20 CODIS loci, while only 35% were obtained using Promega’s PowerPlex® Fusion kit, a current standard CE kit. Mock case-type samples confirmed these results. ForenSeq™ DNA Signature Prep kit demonstrated that it can be successfully used on degraded DNA samples. This study may be helpful for other laboratories assessing and validating MPS technologies.     

Haplotype analysis for Irish ancestry

Forensic haplotype analysis of the male Y chromosome is currently used to establish the number of male donors in sexual assaults, the number of male bleeders in blood pattern analysis, and for ancestry correlation to genetic founder populations in biogeographic studies. In forensic laboratory applications, its primary use is for DNA profile generation with trace amounts of male DNA in the presence of excess female DNA (e.g. spermatozoa identification, male component of fingernail scrapings). Our study supports the potential use of the Y chromosome in a “dragnet” approach (most haplotypes are unique) similar to that described by Kayser in 2017 for solving a cold case sex assault and homicide in The Netherlands. Our study also researched the potential for the identification of an ancestral Irish genetic “footprint” linked to surname O’Brien and identified multiple founder group origins in Ireland and England as well as three samples with the Dal Riata (a Gaelic overkingdom) ancestral haplotype. This study indicates correlation to ancestral Irish ancestry by haplotype but not conclusively to the O’Brien surname.     

DNA分子标记技术在法医植物学中的应用

法医植物学是一门研究与法律事件相关的植物证据的科学。植物物证的DNA分子标记技术是近年来法医植物学研究的主要方向。本文系统地综述了目前应用于法医植物学中的各种DNA分子标记技术，对这些方法的实际应用案例以及可能应用方向进行列举，最后总结出这些分子生物学技术相较于传统植物形态分类方法的优点。     

Comparative study of D1S80 typing by capillary electrophoresis and sequencing

The D1S80 locus is very useful for personal identification in Japan. To obtain a correct allele over 45, we examined PCR amplification product of the allele over 45 both by direct sequencing and fragment analysis using capillary electrophoresis. Direct sequencing finally determined the allele as being 57. However, it was calculated to be an allele of 56 by comparison with size markers for capillary electrophoresis. The difference could be attributed to the electrophoretic size markers. This finding indicates that the direct sequencing may be useful to determine the allele over 45 in the D1S80 locus.     

彗星试验检测死后DNA降解推断PMI的参数选择研究

目的筛选出彗星试验检测死后DNA降解推断死亡时间（PMI）的合适彗星参数。方法大鼠断颈处死后置于10℃和20℃环境中44h，每4h取材脑和骨髓组织制成细胞悬液进行彗星电泳，CASP软件测量彗星参数，线性回归分析和比较各参数与PMI的相关关系。结果死后DNA随PMI的延长逐渐降解，彗星头和尾DNA百分比、尾DNA、Olive尾矩、尾面积、尾矩等参数与PMI呈稳定的线性相关关系。结论彗星头和尾DNA百分比、尾DNA、Olive尾矩、尾面积、尾矩等参数是彗星试验检测死后DNA降解推断PMI的合适参数。     

国产去离子甲酰胺在法医DNA检测中的探讨

目前法医DNA检测中使用的变性辅助溶剂主要是美国AB公司生产的去离子甲酰胺。经调研，本实验选择了目前国内市场上纯度级别最高的实验室用甲酰胺，为AR级。将DNATyper15TM试剂盒分子量内标Typer500分别加入到购买的甲酰胺中，用3130xi遗传分析仪检测，以比较6个厂家生产的甲酰胺对DNA的变性效果，探讨了3家国产去离子甲酰胺用于法医DNA检测上样的可能性。     

Comparison of STR loci profiles obtained from extracted FTA discs using different DNA polymerases

Six commercially available DNA polymerases together with their respective buffers were compared as to their ability to generate profiles of commonly used short tandem repeat loci. Extracted FTA paper discs were used in two fluorescent multiplex PCRs containing the same number of units of DNA polymerase to amplify 21 STR loci and the resultant alleles were visualized after electrophoresis on a capillary sequencer. Significant differences were observed upon comparing the profiles: one polymerase failed to amplify larger alleles and presence of additional peaks varied according to the enzyme used. The use of hot-start polymerases did not affect the quality of the resultant profiles in this comparison.     

A review of low template STR analysis in casework using the DNA SenCE post-PCR purification technique

LGC has developed a method for analysing low-level DNA samples called DNA SenCE (Sensitive Capillary Electrophoresis) based on post-PCR treatment of standard 28-cycle SGMplus PCR product and demonstrated to be equally effective at enhancing profiles as 34-cycle PCR. The method has been validated and accredited and used in casework since July 2007. Inherent in the method is the initial generation of a standard 28-cycle SGMplus profile so a direct comparison of standard and DNA SenCE results for all casework is possible. Here we review DNA SenCE casework, reporting the magnitude of peak enhancement and stochastic effects seen in the DNA SenCE profiles.     

用非放射性标记MYO探针进行DNA指纹分析

用非放射性标记ＭＹＯ探针进行人的ＤＮＡ指纹分析，获得了清晰易读的ＤＮＡ指纹图，并用该方法调查了云南省的７８名无关个体，经统计学分析，计算出无关个体的相关几率是３．４×１０－１０，高于３２Ｐ标记的Ｍｙｏ探针。研究结果表明该方法是一种简便、快速、灵敏、可靠、经济的ＤＮＡ指纹图检测技术，可在法医鉴定及其它领域里得到更广泛的应用。     

A case of tri-allelic pattern at locus D3S1358 on chromosome 3p21 inherited from paternal grandmother

We are reporting a case of tri-allelic inheritance at locus D3S1358 commonly used for genetic identification in forensic DNA testing. This case was encountered during routine paternity testing using commercial DNA profiling kits. The tri-allelic inheritance identified was probably a result of duplication at this locus, supported by the equal peak intensities and inheritance pattern from grandparent to child.