Application of mtDNA sequence analysis in forensic casework for the identification of human remains

In four forensic cases of unidentified skeletal remains investigated in the last year, we were able to attach three to missing persons. In one case we could show that the discovered bone sample did not fit to a missing child. The method for mitochondrial DNA analysis for the routine identification of skeletal remains was established in our institute by typing bone samples of defined age obtained from Frankfurt's cemetery. Reproducible results were obtained for bones up to 75 years old. For analysis the bone samples were pulverised to fine powder, decalcified and DNA was extracted. From the DNA we amplified a 404-bp fragment from HV-1 and a 379-bp fragment from HV-2 of the mtDNA control region. After sequencing of the PCR products, the results were compared to the Anderson reference sequence and to putative maternal relatives.     

A method to enable forensic genetic genealogy investigations from DNA mixtures

The presence of more than one DNA contributor in an evidentiary sample may preclude attempts to use forensic genetic genealogy to develop an investigative lead. To address this issue, we developed a workflow for deconvolution of SNP mixtures into single source profiles that are suitable for matching against a genealogical database. Using the method, two-contributor DNA mixtures assayed using a commercial SNP typing kit can produce informative match results for both major and minor contributors.     

An improved method for post-PCR purification for mtDNA sequence analysis

Mitochondrial DNA (mtDNA) analysis of forensic samples typically is performed when the quantity and quality of DNA are insufficient for nuclear DNA analysis or when maternal relatives may be the only reference source. Many of the steps required in the analytical process are both lengthy and labor intensive. Therefore, improvements in the process that reduce labor without compromising the quality of the data are desirable. The current procedure requires purification of the amplicons by centrifugal filtration after PCR and prior to cycle sequencing. Because this method requires several manipulations to perform, alternate cleanup procedures were investigated. These include the use of 1) Qiagen QlAquick PCR Purification columns, 2) Concert Rapid PCR Purification columns, and 3) ExoSAP-IT reagent. When the yield of purified amplicons, quality of the sequence profile, and ease of assay were evaluated, the use of ExoSAP-IT reagent for post-amplification purification was chosen to replace the filtration method.     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Application of mtDNA SNP analysis in forensic casework

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.     

A PCR multiplex and database for forensic DNA identification of dogs

Animal-derived trace evidence is a common finding at crime scenes and may provide an important link between victim(s) and suspect(s). A database of 558 dogs of pure and mixed breeds is described and analyzed with two PCR multiplexes of 17 microsatellites. Summary statistics (number of alleles, expected and observed heterozygosity and power of exclusion) are compared between breeds. Marked population substructure in dog breeds indicates significant inbreeding, and the use of a conservative theta value is recommended in likelihood calculations for determining the significance of a DNA match. Evidence is presented that the informativeness of the canine microsatellites, despite inbreeding, is comparable to the human CODIS loci. Two cases utilizing canine DNA typing, State of Washington v. Kenneth Leuluaialii and George Tuilefano and Crown v. Daniel McGowan, illustrate the potential of canine microsatellite markers for forensic investigations.     

A DNA microarray system for forensic SNP analysis

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.     

A call for mtDNA data quality control in forensic science

There is increasing evidence that many of the mitochondrial DNA (mtDNA) databases published in the fields of forensic science and molecular anthropology are flawed. An a posteriori phylogenetic analysis of the sequences could help to eliminate most of the errors and thus greatly improve data quality. However, previously published caveats and recommendations along these lines were not yet picked up by all researchers. Here we call for stringent quality control of mtDNA data by haplogroup-directed database comparisons. We take some problematic databases of East Asian mtDNAs, published in the Journal of Forensic Sciences and Forensic Science International, as examples to demonstrate the process of pinpointing obvious errors. Our results show that data sets are not only notoriously plagued by base shifts and artificial recombination but also by lab-specific phantom mutations, especially in the second hypervariable region (HVR-II).     

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.     

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。     

A novel method of automated skull registration for forensic facial approximation

Modern forensic facial reconstruction techniques are based on an understanding of skeletal variation and tissue depths. These techniques rely upon a skilled practitioner interpreting limited data. To (i) increase the amount of data available and (ii) lessen the subjective interpretation, we use medical imaging and statistical techniques. We introduce a software tool, reality enhancement/facial approximation by computational estimation (RE/FACE) for computer-based forensic facial reconstruction. The tool applies innovative computer-based techniques to a database of human head computed tomography (CT) scans in order to derive a statistical approximation of the soft tissue structure of a questioned skull. A core component of this tool is an algorithm for removing the variation in facial structure due to skeletal variation. This method uses models derived from the CT scans and does not require manual measurement or placement of landmarks. It does not require tissue-depth tables, can be tailored to specific racial categories by adding CT scans, and removes much of the subjectivity of manual reconstructions.     

DNA甲基化在法医DNA分析中的应用

表观遗传学在生命的发生、发展过程中起着十分重要的作用。DNA甲基化作为表观遗传的一个重要方面,不仅参与多种基因的表达调控,与机体的发育、肿瘤发生等密切相关,而且具有可遗传性、相对稳定性、亲缘特异性、基因组中含量丰富等特点,已证实适用于法医DNA分析。本文对近年来DNA甲基化在印迹基因、同卵双生子鉴定、年龄、性别推断方面的研究与应用进行回顾与综述,以期为在法医学及相关领域中应用提供参考。     

Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.     

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。     

国产磁珠Wawasye试剂盒在法医DNA中应用

目的探讨国产磁珠Wawasye试剂盒在法医DNA检案中的应用效果。方法利用475份各类常见生物检材测试其效果,并与M48磁珠试剂盒作比较。结果国产磁珠Wawasye试剂盒与M48磁珠试剂盒在检验各类常见生物检材的结果无显著性差异。结论国产磁珠Wawasye试剂盒适用于公安机关DNA实验室。     

Choice of population database for forensic DNA profile analysis

When evaluating the weight of evidence (WoE) for an individual to be a contributor to a DNA sample, an allele frequency database is required. The allele frequencies are needed to inform about genotype probabilities for unknown contributors of DNA to the sample. Typically databases are available from several populations, and a common practice is to evaluate the WoE using each available database for each unknown contributor. Often the most conservative WoE (most favourable to the defence) is the one reported to the court. However the number of human populations that could be considered is essentially unlimited and the number of contributors to a sample can be large, making it impractical to perform every possible WoE calculation, particularly for complex crime scene profiles. We propose instead the use of only the database that best matches the ancestry of the queried contributor, together with a substantial F_ST adjustment. To investigate the degree of conservativeness of this approach, we performed extensive simulations of one- and two-contributor crime scene profiles, in the latter case with, and without, the profile of the second contributor available for the analysis. The genotypes were simulated using five population databases, which were also available for the analysis, and evaluations of WoE using our heuristic rule were compared with several alternative calculations using different databases. Using F_ST = 0.03, we found that our heuristic gave WoE more favourable to the defence than alternative calculations in well over 99% of the comparisons we considered; on average the difference in WoE was just under 0.2 bans (orders of magnitude) per locus. The degree of conservativeness of the heuristic rule can be adjusted through the F_ST value. We propose the use of this heuristic for DNA profile WoE calculations, due to its ease of implementation, and efficient use of the evidence while allowing a flexible degree of conservativeness.     

A comparison of statistical models for the analysis of complex forensic DNA profiles

Complex mixtures and LtDNA profiles are difficult to interpret. As yet there is no consensus within the forensic biology community as to how these profiles should be interpreted. This paper is a review of some of the current interpretation models, highlighting their weaknesses and strengths. It also discusses what a forensic biologist requires in an interpretation model and if this can be realistically executed under current justice systems.     

Significance of mitochondrial DNA for forensic stain analysis, identification and forensic lineage determination

DNA testing using conventional STR systems may produce insufficient results, if the genomic DNA in the specimen is either highly degraded or the available quantity is very small (e.g. skin particles, hair shafts or ancient bones). In some of these cases the examination of mitochondrial DNA, which is present in considerably larger copy numbers in the cytoplasm, is more successful than that of nuclear DNA. Identification of unknown corpses by conventional DNA typing sometimes remains doubtful, if only samples from presumably distant relatives or putative brothers or sisters are available for comparison. Since mitochondrial DNA is generally transmitted in maternal lineages, its sequence pattern can be directly compared with those of other individuals and, in case of the same maternal lineage, corresponding sequence chromatograms are to be expected. In connection with nuclear DNA typing methods certain sequence motives may furnish clues to ethnic groups. The report presents three cases illustrating the application possibilities of mtDNA typing in forensic practice.     

A novel method for STR-based DNA profiling using microarrays

We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.