Completely automated interpretation of reference samples

Inspired by fully continuous interpretation a completely automated interpretation workflow for reference samples was implemented in our lab. It's based on a quite simple model, which includes DNA amount and degradation as well as backward and forward stutters. The current implementation can handle CE results of any number of replicates, even from different autosomal or Y-chromosomal STR kits. Results, lessons learned in a first set of test series and potential future extensions are discussed.     

Subjectivity and bias in forensic DNA mixture interpretation

The objectivity of forensic science decision making has received increased attention and scrutiny. However, there are only a few published studies experimentally addressing the potential for contextual bias. Because of the esteem of DNA evidence, it is important to study and assess the impact of subjectivity and bias on DNA mixture interpretation. The study reported here presents empirical data suggesting that DNA mixture interpretation is subjective. When 17 North American expert DNA examiners were asked for their interpretation of data from an adjudicated criminal case in that jurisdiction, they produced inconsistent interpretations. Furthermore, the majority of 'context free' experts disagreed with the laboratory's pre-trial conclusions, suggesting that the extraneous context of the criminal case may have influenced the interpretation of the DNA evidence, thereby showing a biasing effect of contextual information in DNA mixture interpretation.     

Probabilistic genotyping of single cell replicates from complex DNA mixtures recovers higher contributor LRs than standard analysis

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2–6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥10²⁰) from donors that had returned non-probative LRs (<10³) by standard methods.     

DNA分析技术在法科学中的应用及展望

本综述对DNA指纹、PCR -VNTR、PCR -STR、PCR -mtDNA测序等技术的发展 ,以及其在法科学中的应用领域和发展前景作了系统的阐述。认为由于DNA分析技术所具有的特点 ,使之已成为现今法科学生物检材检验的主要手段之一。阐述了现阶段DNA分析技术已向标准化、自动化和高鉴别机率方向发展 ,以及建立DNA罪犯数据库的必要性和应用价值     

Using previous DNA casework data to aid decision making in the process of DNA profile interpretation

In this study, a DNA decision support tool was developed. With this tool we aim to gain insight in what actions are performed with which (types of) DNA profiles in casework, to improve decision making by DNA experts in future cases.     

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.     

中国法庭科学DNA数据库

本文综述了中国法庭科学DNA数据库的建立和发展过程、DNA数据库结构、内容、特点、作用以及存在的问题、发展方向、展望,目的是为如何进一步建设好具有中国特色的DNA数据库提供借鉴。     

Identification and separation of DNA mixtures using peak area information

We introduce a new methodology, based upon probabilistic expert systems, for analysing forensic identification problems involving DNA mixture traces using quantitative peak area information. Peak area is modelled with conditional Gaussian distributions. The expert system can be used for ascertaining whether individuals, whose profiles have been measured, have contributed to the mixture. It can also be used to predict DNA profiles of unknown contributors by separating the mixture into its individual components. The potential of our probabilistic methodology is illustrated on case data examples and compared with alternative approaches. The advantages are that identification and separation issues can be handled in a unified way within a single probabilistic model and the uncertainty associated with the analysis is quantified. Further work, required to bring the methodology to a point where it could be applied to the routine analysis of casework, is discussed.     

Qubit~快速荧光定量系统在法医学中的应用

目的研究Qubit定量系统在法医学中的应用。方法使用Quant-iTTM试剂盒检测法医DNA样本并得出样本浓度。结果Quant-iTTM试剂盒能检测并定量各种生物学检材DNA样本。结论Quant-iTTM试剂盒能够应用在法医学检验中。     

The use of wildcards in forensic DNA database searches

Forensic genetic databases contain millions of DNA profiles worldwide. An allelic drop-out at a heterozygous locus (two different alleles) erroneously results in an observed homozygous profile. To guard against this, wildcards are induced for homozygous profiles. The wildcard represents any allele including the observed allele.In this paper, theoretical expressions for the number of matching loci are derived, results shown from a Danish dataset with more than 50,000 DNA profiles and simulations used to link homozygosity with the drop-out probability, which best mimics wildcard matching.     

单核苷酸多态性与法医学DNA分型技术

单核苷酸多态性(single nucleotide polymorphism,SNP)分型技术越来越成为法医学领域关注的热点,它在研究Y染色体或线粒体单倍型以及DNA表型的分析中具有重要应用价值。本文着重比较分析了SNP技术与片段长度多态性技术之间的优劣,同时就当前STR位点识别概率与所需选择的SNP位点数进行探讨。此外,本文还就各类SNP分型方法的优缺点及其法医学应用进行了论述。     

Quadruplex real-time PCR for forensic DNA quantitation

Forensic DNA quantitation is an important initial step preceding PCR amplification of the STR loci even though information concerning the quality of the DNA is not revealed. A quadruplex real-time PCR (qPCR) assay was developed to quantify four DNA targets: (1) the human RB1 gene in nuclear DNA, (2) the DAZ gene present on the human Y chromosome, (3) the ATPase8 gene present in human mitochondrial DNA and (4) an artificial internal positive control to reveal possible PCR inhibition. Primers labeled with four different fluorophores are used together with a single quencher using the antiprimer quenching-based qPCR method in one reaction, in which the resultant amplicons are less than 127 bp in size. Sensitivity was shown to be less than ten copies for all four targets in the absence of amplification inhibition. The amplification remained sensitive in the presence of an excess of non-human DNA.     

A fast analysis system for forensic DNA reference samples

On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a new analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA^® card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog.     

Application of BioRobot M48 to forensic DNA extraction

The development of a nucleic acid extraction method based on magnetic separation has opened up possibilities forl automation of DNA extraction. The BioRobot M48 is one of robotic stations applicable to automated DNA extraction in forensics. However, each new method should be thoroughly validated before application to routine casework. Our aim was to compare the effectiveness of the currently utilized organic/Microcon 100 based extraction procedure and magnetic extraction with BioRobot M48. The DNA concentration of DNA extracts obtained from different kinds of typical forensic material was evaluated followed by amplification with the SGM Plus or Identifiler kit and capillary electrophoresis using ABI 3100 Avant. We can conclude that BioRobot M48 is a very effective instrument for DNA extraction from most specimens and can be successfully applied in forensic laboratories.     

Use of subpopulation data in Australian forensic DNA casework

DNA profiling evidence presented in court should be accompanied by a reliable estimate of its evidential weight. In calculating such statistics, allele frequencies from commonly employed autosomal microsatellite loci are required. These allele frequencies should be collected at a level that appropriately represents the genetic diversity that exists in the population. Typically this occurs at broadly defined bio-geographic categories, such as Caucasian or Asian. Datasets are commonly administered at the jurisdictional level. This paper focuses on Australian jurisdictions and assesses whether this current practice is appropriate for Aboriginal Australian and Caucasian populations alike. In keeping with other studies we observe negligible differences between Caucasian populations within Australia when segregated geographically. However segregation of Aboriginal Australian population data along contemporary State and Territory lines appears to mask the diversity that exists within this subpopulation. For this reason datasets collated along more traditional lines may be more appropriate, particularly to distinguish the most genetically differentiated populations residing in the north of the continent.     

Evaluating probabilistic genotyping for low-pass DNA sequencing

Most genomic methods consider the sample genotype. Data are evaluated at some location, and if the signal strength is sufficient, a genotype call is made. Conversely, sites that lack sufficient signal are treated as missing data. Such methods for genotype calling are binary, and this dichotomy limits genomic analyses to relatively high-coverage (and high-cost) massively parallel sequencing (MPS) data. It follows that bioinformatic methods that rely on genotypes may not be ideal for trace DNA samples, such as those sometimes encountered in forensic investigations, but even when applicable such analyses can be expensive. However, there are some genomic analyses where having many uncertain genotypes (with measured uncertainty) assayed over the entirety of the genome may be more powerful than current multi-locus approaches that consider a limited number of well-characterized markers. Methods for such problems may rely on genotype likelihood, which expresses the likelihood of alternative genotype calls in addition to the most likely call. One application that can benefit from genotype likelihoods is kinship analysis. NgsRelate is a bioinformatic tool that infers pairwise relatedness using a probabilistic genotyping framework, which accommodates the uncertainty associated with genotype calls for low-pass MPS data. Here, NgsRelate was used to infer kinship coefficients from low-pass whole genome sequencing data from a known pedigree. Multiple samples in a titration series (ranging from 50 ng to 0.5 ng) on a single MPS S4 flow cell were assessed. A reproducible scientific bioinformatic workflow was developed to evaluate kinship coefficients considering up to 3rd degree relatives. NgsRelate was found to provide robust assessments of kinship. Further, the use of low-pass MPS data provides a more cost-effective way to conduct forensic investigations.     

中国法医学会物证专业委员会法医DNA分析的若干建议

中国法医学会法医物证学专业委员会与国际法医遗传学会中文专委会于2006年10月在成都召开学术会议。我们的讨论强调有必要将国际法医遗传学会的信息及时传递到中国。因此,按照国际法医遗传学会的指南,我们推荐混合斑分析,法医DNA数据库及新遗传标记选择标准供同行参考。     

用熔解曲线法分析插入/缺失多态性和Y染色体SNPs多态性

随着单核苷酸多态性SNPs (SingleNucleotidePolymorphisms)及插入 /缺失多态性Indels (Insertion/Dele tion)的分型技术研究的深入 ,SNPs和Indels在法医学上的应用将受到深刻的影响。本文研究和探讨Indels的分型方法 ,通过测定扩增DNA片段在溶液中的溶解曲线图确定每个样品的基因型 ,称为溶解曲线Indels基因分型方法(McI/D)。溶解曲线图由被测样品DNA片段的特殊溶解温度组成 ,扩增结果直接由仪器分析不需要繁杂的PCR后期操作。     

人线粒体DNA序列分析在法医学中的应用研究及其进展

综述人线粒体DNA(m tDNA)序列分析在法医学种属鉴别、个体识别,以及个体年龄推断中的应用研究及其进展,展望对m tDNA异质性的研究及建立人m tDNA数据库,并具有重要的法医学实践意义。     

人类线粒体DNA异质性研究进展及在法医学中的应用

线粒体DNA(mtDNA)异质性的存在使其在法医学应用变得复杂。本文对mtDNA异质性形成的可能原因、异质性的分布和遗传特点、异质性的筛查和定量方法、异质性对法医学的影响以及异质性的研究和展望等方面进行综述,探讨异质性在法医学上的应用价值。