DNA profiling of disaster victim identification in Trenggalek shipwreck case

DNA analysis is one of the primary methods of identification in DVI practices. The external environment of a mass disaster often results in severe fragmentation, decomposition and intermixing of the remains. However, DNA profiling still can be achieved even on cases involving partial, severely decomposed remains. This report shows the DNA profile of shipwreck victims using identifiler plus marker from tissue sample exposed to environmental conditions.     

The Ge.F.I. DNA Proficiency Test: Year-one experience

The Ge.F.I. (the Italian Speaking Working Group of ISFG) has launched the first DNA Proficiency Test in 2012. The aim is to increase the external quality controls in forensic genetics in Italy and to drive to the standardizations of methods within the laboratories. Reference and mixed stains typing as well as statistical exercises were proposed and 26 laboratories submitted results.     

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.     

A 1-year time course study of human RNA degradation in body fluids under dry and humid environmental conditions

Blood, saliva and semen are some of the forensically most relevant biological stains found at crime scenes. mRNA profiling is a reliable approach for the identification of the origin of an evidentiary trace. A stable set of markers and the knowledge about the effects of RNA degradation under different environmental conditions is necessary for the determination of an unknown biological stain. The aim of this work was to compare RNA degradation for human blood, semen and saliva at three different concentrations during a 1-year time period and exposed to dry and humid conditions. Also, this study addressed the question whether there are relevant differences in the efficiency of two RNA extraction methods.     

Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification

Traditional body fluid identification methods use a variety of technologically diverse techniques that do not permit the identification of all body fluids. Definitive identification of the biological material present can be crucial to a fuller understanding of the circumstances pertaining to a crime. Thus definitive molecular based strategies for the conclusive identification of forensically relevant biological fluids need to be developed. Messenger (mRNA) profiling is an example of such a molecular based approach.Current mRNA body fluid identification assays typically involve either capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed multiplex high resolution melt (HRM) assays that provide an identification of all forensically relevant biological fluids and tissues.     

The efficiency of Y-chromosome markers in forensic trace analysis and their inclusion in the Austrian National DNA Database

Y-STR markers are a valuable tool in the analysis of biological traces in which a mixture of male and female trace material is to be expected. It is possible to generate a Y-chromosome DNA profile, even if all the prior sperm tests are negative and no sign of any male component is found in amelogenin. In 38 of a total of 239 sexual offences a perpetrator trace was identified solely using Y-STR analysis. Based on these findings, the Austrian National DNA Database was expanded to include Y-STRs in 2012 with the primary objective to identify serial sexual offences.     

Evaluation of five DNA extraction systems for recovery of DNA from bone

Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch^® gDNA Plant Kit (Life Technologies), DNA IQ^TM System Kit (Promega), DNeasy^® Blood & Tissue Kit (Qiagen) and PrepFiler^® BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq^® qPCR Master Mix (Promega) using an Applied Biosystems^® 7500 Real-Time PCR System and the extracts were amplified using an in-house multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch^® gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition.     

SNP genotyping of forensic casework samples using the 52 SNPforID markers

The analysis of degraded DNA is one of the biggest challenges in forensic casework. SNPs, which can be amplified using small amplicons, have previously been successfully applied to the profiling of forensic evidence that could not be analyzed using conventional STRs. Here we selected the 52 SNPforID SNP markers, with amplicons that ranged in size from 59 bp to 115 bp, and used them to profile a range of casework samples from Malaysia. DNA degradation is a common problem in Malaysia due to the high temperatures and humidity. To carry out the study we modified the 52 SNPforID markers into four 13-plex SNaPshot assays to enable easier interpretation of profiles on the ABI PRISM^® 310 and 3500.Fifty-one crime samples comprising bloodstains on cloth, swabs, and a mat and 2 swabs of trace DNA from 10 crime scenes in Malaysia were profiled after DNA extraction using a phenol–chloroform method. The samples were also subjected to STR analysis using the Powerplex^® 16 system (Promega), which resulted in only 17 full profiles and 9 partial profiles; using SNPs, 36 full profiles and 5 partial profiles could be generated.     

mtDNAprofiler: A Web Application for the Nomenclature and Comparison of Human Mitochondrial DNA Sequences,

Mitochondrial DNA (mtDNA) is a valuable tool in the fields of forensic, population, and medical genetics. However, recording and comparing mtDNA control region or entire genome sequences would be difficult if researchers are not familiar with mtDNA nomenclature conventions. Therefore, mtDNAprofiler, a Web application, was designed for the analysis and comparison of mtDNA sequences in a string format or as a list of mtDNA single‐nucleotide polymorphisms (mtSNPs). mtDNAprofiler which comprises four mtDNA sequence‐analysis tools (mtDNA nomenclature, mtDNA assembly, mtSNP conversion, and mtSNP concordance‐check) supports not only the accurate analysis of mtDNA sequences via an automated nomenclature function, but also consistent management of mtSNP data via direct comparison and validity‐check functions. Since mtDNAprofiler consists of four tools that are associated with key steps of mtDNA sequence analysis, mtDNAprofiler will be helpful for researchers working with mtDNA. mtDNAprofiler is freely available at http://mtprofiler.yonsei.ac.kr .     

A Comparison of Enhancement Techniques for Footwear Impressions on Dark and Patterned Fabrics,

The use of chemical enhancement techniques on porous substrates, such as fabrics, poses several challenges predominantly due to the occurrence of background staining and diffusion as well as visualization difficulties. A range of readily available chemical and lighting techniques were utilized to enhance footwear impressions made in blood, soil, and urine on dark and patterned fabrics. Footwear impressions were all prepared at a set force using a specifically built footwear rig. In most cases, results demonstrated that fluorescent chemical techniques were required for visualization as nonfluorescent techniques provided little or no contrast with the background. Occasionally, this contrast was improved by oblique lighting. Successful results were obtained for the enhancement of footwear impressions in blood; however, the enhancement of footwear impressions in urine and soil on dark and patterned fabrics was much more limited. The results demonstrate that visualization and fluorescent enhancement on porous substrates such as fabrics is possible.