Integrated multidisciplinary approach in a case of occupation related planned complex suicide-peticide

The combined event of a suicide and the killing of a pet has been hardly explored in forensic literature, but it is not rare at all. In the reported case the dead corpse of a 60-year old mentally ill woman and the carcass of a dog were found on the bed of a private apartment. In light of death scene investigation, necroscopic examination and toxicological analyses death was attributed to licit drug intoxication and self-strangulation after lethal poisoning of the dog. Due to the presence of two lethal means (cervical noose and drugs), acting in chronological order, the event was classified as a secondary complex suicide. Moreover, the woman, although mentally ill, was a psychiatrist. Thus, her professional background, namely the easy access to psychiatric drugs together with her knowledge of drug composition and properties lead to suppose an occupation related suicide. The comprehensive analysis of all the available information, including death scene investigation, occupational, necroscopic and toxicological data, resulted of the utmost importance for a proper reconstruction of the events and are recommended in complex cases such as occupation related planned complex suicides combined to the killing of pets.     

Identifying muscle hemorrhage in rat cadavers with advanced decomposition by FT-IR microspectroscopy combined with chemometrics

The identification of muscle hemorrhage in a cadaver that is in an advanced stage of decomposition is an important but challenging task. Our study investigated whether Fourier transform infrared (FT-IR) microspectroscopy in conjunction with chemometrics could identify muscle hemorrhage using rat cadavers with advanced decomposition. In this study, an intramuscular blood injection method, instead of a mechanical injury method, was used to construct a muscle hemorrhage model, and the modeling idea of muscle hemorrhage identification was to discriminate and classify hemoglobin-leaking myofibrils from negative myofibrils. First, the optical images of hematoxylin/eosin (H&E) stained hemorrhagic muscle at different postmortem intervals (PMIs) were observed and showed that the morphological features of whole erythrocytes disappeared since the PMI of 4 d. Subsequently, principle component analysis (PCA) was performed and indicated that the biochemical differences in protein structures between fresh erythrocytes and myofibrils can be detected by the IR spectroscopic method. Ultimately, several classification models based on the partial least square discriminant analysis (PLS-DA) algorithm were successfully constructed for different PMIs and PMI ranges and achieved great prediction performances in external validations. This preliminary study demonstrates the feasibility of using FT-IR microspectroscopy combined with chemometrics as a potential approach for identifying muscle hemorrhage in cadavers with advanced decomposition for offering vital evidences in judicial process.     

Evaluation of marker selection methods and statistical models for chronological age prediction based on DNA methylation

In forensic investigation, retrieving biological information from DNA evidence is a promising field of interest. One of the applications is on the estimation of the age of the donor based on DNA methylation. A large number of studies focused on age prediction using the 450 K Human Methylation Beadchip. Various marker selection methods and prediction models have been considered. However, there is a lack of research evaluating different high-dimensional variable selection methods of CpG sites with various models for age prediction. The aim of this study is to evaluate four variable selection methods (forward selection, LASSO, elastic net and SCAD) combined with a classical statistical model and sophisticated machine learning models based on the mean absolute deviation (MAD) and the root-mean-square error (RMSE). We used publicly available 450 K data set containing 991 whole blood samples (age 19–101 years). We found that the multiple linear regression model with 16 markers selected from the forward selection method performed very well in age prediction (MAD = 3.76 years and RMSE = 5.01 years). On the other hand, the highly advanced ultrahigh dimensional variable selection methods and sophisticated machine learning algorithms appeared unnecessary for age prediction based on DNA methylation.     

Applicability of the SNPforID 52-plex panel for human identification and ancestry evaluation in a Brazilian population sample by next-generation sequencing

SNP analysis is of paramount importance in forensic genetics. The development of new technologies in next-generation sequencing allowed processing a large number of markers in various samples simultaneously. Although SNPs are less informative than STRs, they present lower mutation rates and perform better when using degraded samples. Some SNP systems were developed for forensic usage, such as the SNPforID 52-plex, from the SNPforID Consortium, containing 52 bi-allelic SNPs for human identification. In this paper we evaluated the informativeness of this system in a Brazilian population sample (n = 340). DNA libraries were prepared using a customized HaloPlex Target Enrichment System kit (Agilent Technologies, Inc.) and sequenced in the MiSeq Personal Sequencer platform (Illumina Inc.). The methodology presented here allowed the analysis of 51 out of 52 SNPforID markers. Allele frequencies and forensic parameters were estimated, revealing high informativeness: the combined match probability and power of exclusion were 6.48 × 10⁻²¹ and 0.9997, respectively. Population admixture analysis indicates high European contribution (more than 70%) and low Amerindian contribution (less than 10%) in our population, while individual admixture analyses were consistent with the majority of individuals presenting high European contribution. This study demonstrates that the 52-plex kit is suitable for forensic cases in a Brazilian population, presenting results comparable with those obtained using a 16 STR panel.     

Forensic Y-SNP analysis beyond SNaPshot: High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing

Y-chromosomal haplogroups assigned from male-specific Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal bio-geographic ancestry inference, both being relevant in forensic genetics. However, most previously developed forensic Y-SNP tools did not provide Y haplogroup resolution on the high level needed in forensic applications, because the limited multiplex capacity of the DNA technologies used only allowed the inclusion of a relatively small number of Y-SNPs. In a proof-of-principle study, we recently demonstrated that high-resolution Y haplogrouping is feasible via two AmpliSeq PCR analyses and simultaneous massively parallel sequencing (MPS) of 530 Y-SNPs allowing the inference of 432 Y-haplogroups. With the current study, we present a largely improved Y-SNP MPS lab tool that we specifically designed for the analysis of low quality and quantity DNA often confronted with in forensic DNA analysis. Improvements include i) Y-SNP marker selection based on the “minimal reference phylogeny for the human Y chromosome” (PhyloTree Y), ii) strong increase of the number of targeted Y-SNPs allowing many more Y haplogroups to be inferred, iii) focus on short amplicon length enabling successful analysis of degraded DNA, and iv) combination of all amplicons in a single AmpliSeq PCR and simultaneous sequencing allowing single DNA aliquot use. This new MPS tool simultaneously analyses 859 Y-SNPs and allows inferring 640 Y haplogroups. Preliminary forensic developmental validation testing revealed that this tool performs highly accurate, is sensitive and robust. We also provide a revised software tool for analysing the sequencing data produced by the new MPS lab tool including final Y haplogroup assignment. We envision the tools introduced here for high-resolution Y-chromosomal haplogrouping to determine a man’s paternal lineage and/or paternal bio-geographic ancestry to become widely used in forensic Y-chromosome DNA analysis and other applications were Y haplogroup information from low quality / quantity DNA samples is required.     

Pilot study for forensic evaluations of the Precision ID GlobalFiler™ NGS STR Panel v2 with the Ion S5™ system

With the continuous development of massively parallel sequencing (MPS), increasing numbers of laboratories have utilized this method for forensic genomic analyses. When sequencing common short tandem repeats (STRs), MPS does have many advantages over the length-based genotyping method that uses traditional capillary electrophoresis (CE) technology. The Precision ID GlobalFiler™ NGS STR Panel v2 was recently released to simultaneously target 31 autosomal STRs (20 expanded Combined DNA Index System (CODIS) core loci and 11 non-CODIS loci) and 4 gender determination loci (Amelogenin, DYS391, SRY and Y-indel (rs2032678)) with the Ion S5™ System. In the current study, we performed a preliminary validation for this novel MPS-STR panel that included the following analyses: repeatability, concordance, stutter and balance, sensitivity, case-type sample testing, stability, mixture and a population investigation. Complete and reliable profiles were obtained using 125 pg of positive control DNA. The commonly encountered types of case samples and artificial mixtures with ratios of 1:1, 1:3 and 3:1 were also fully genotyped. Additional allele sequence variations were detected in samples from 50 unrelated individuals, and subsequently, an increased power of discrimination and power of exclusion were achieved. However, the average depth of coverage (DoC) of the Penta D locus was detected to be dramatically lower than those of other loci, which caused an interlocus imbalance; this could be one of the reasons for the intralocus imbalance of this locus and the 0.18% inconsistent results in the concordance study. Although certain flaws were observed, the informative metrics, including the DoC, sequence coverage ratios (SCRs) and heterozygote balance (H_b), of the novel MPS multiplex in our study were sufficient for reliable sequencing results that were 99.61% in concordance with the capillary electrophoresis (CE) results. In general, the Precision ID GlobalFiler™ NGS STR Panel v2 was demonstrated to be sensitive, reliable and robust and could be a powerful tool for human identification and kinship analyses. Additionally, we look forward to its updated version.     

Short tandem repeat typing on the 454 platform: Strategies and considerations for targeted sequencing of common forensic markers

To investigate the feasibility of next generation sequencing technology (NGS) for the multiplex detection and sequence production of short tandem repeats (STRs) from degraded and low DNA quantity samples, standard polymerase chain reaction amplification methods were used to enrich for commonly employed STR markers. Samples were amplified with two multiplexing strategies: a multiplex containing thirteen miniSTR markers and a series of multiplexes containing four miniSTR markers each. Each sample multiplex was barcoded with a sample-specific multiplex identifier for subsequent parallel tagged sequencing on the GS Junior System (454 Life Sciences, a Roche company, Branford, CT). Sequencing results from over fifty DNA extracts representing both pristine samples and low-quality evidentiary specimens reflected known genotypes and were consistent across multiple extracts and/or amplifications of the same sample. Furthermore, the NGS data revealed sequence information not available with standard capillary electrophoresis-based detection alone. For the population samples tested, a total of 152 single nucleotide polymorphisms or insertions/deletions were identified in over 935 recovered alleles, averaging one polymorphism for every six recovered alleles. For three of the loci, the sequence information doubled the number of alleles detected via traditional STR typing by fragment analysis. In addition, twenty-eight of these variants were only seen once within our dataset, highlighting the potential for discrimination among individuals. These additional data are likely to be particularly valuable in missing persons and disaster victim identification cases for which only partial profiles may be recovered and/or only distant kin are available as references. And, considering the opportunity to target only small amplicons with NGS, this type of STR typing will allow for greater information recovery from challenging casework samples. While our results highlight the potential of new technologies for recovering discriminatory genetic information from evidentiary specimens, our data also reveal the complexities of NGS-based STR typing, both in terms of the laboratory assays themselves as well as the downstream data processing and analysis.     

Haplotype diversity of 17 Y-chromosome STR loci in Han population from different areas of Sichuan Province,Southwest China

The distribution of 17 Y-chromosome short tandem repeat (STR) loci, included in the AmpFlSTR®Yfiler™ amplification kit, were analyzed in six different samplings (N = 878) from Sichuan, China. Haplotype diversity and discrimination capacity (DC) values were calculated. Pairwise Rst values were evaluated in AMOVA analysis and visualized through multidimensional scaling (MDS). A total of 547 unique haplotypes were detected. The observed haplotype diversity and discrimination capacity were 0.9995 and 0.7745, respectively. The homogeneity of Sichuan Han population was detected when microareas were analyzed. This population exhibited no significant genetic difference to both of the minorities in reference databases, Mongolian and Manchu, which had been through mass ethnic amalgamation with Sichuan Han population in history.     

Population and performance analyses of four major populations with Illumina’s FGx Forensic Genomics System

The MiSeq FGx Forensic Genomics System (Illumina) enables amplification and massively parallel sequencing of 59 STRs, 94 identity informative SNPs, 54 ancestry informative SNPs, and 24 phenotypic informative SNPs. Allele frequency and population statistics data were generated for the 172 SNP loci included in this panel on four major population groups (Chinese, African Americans, US Caucasians, and Southwest Hispanics). Single-locus and combined random match probability values were generated for the identity informative SNPs. The average combined STR and identity informative SNP random match probabilities (assuming independence) across all four populations were 1.75E-67 and 2.30E-71 with length-based and sequence-based STR alleles, respectively. Ancestry and phenotype predictions were obtained using the ForenSeq™ Universal Analysis System (UAS; Illumina) based on the ancestry informative and phenotype informative SNP profiles generated for each sample. Additionally, performance metrics, including profile completeness, read depth, relative locus performance, and allele coverage ratios, were evaluated and detailed for the 725 samples included in this study. While some genetic markers included in this panel performed notably better than others, performance across populations was generally consistent. The performance and population data included in this study support that accurate and reliable profiles were generated and provide valuable background information for laboratories considering internal validation studies and implementation.