RESUMEN
The forensic science community is poised to utilize modern advances in massively parallel sequencing (MPS) technologies to better characterize biological samples with higher resolution. A critical component towards the advancement of forensic DNA analysis with these technologies is a comprehensive understanding of the diversity and population distribution of sequence-based short tandem repeat (STR) alleles. Here we analyzed 786 samples of individuals from different population groups, including four of the mostly commonly encountered in forensic casework in the USA. DNA samples were ampliï¬ed with the PowerSeq™ Auto/Y System Prototype Kit (Promega Corp.), and sequencing was performed on an Illumina® MiSeq instrument. Sequence data were analyzed using a bioinformatics processing tool, Altius. For additional data analysis and profile comparison, capillary electrophoresis (CE) size-based STR genotypes were generated for a subset of individuals, and where possible, also with a second commercially available MPS STR assay. Autosomal STR loci were analyzed and frequencies were calculated based on sequence composition. Also, population genetics studies were performed, with Hardy-Weinberg equilibrium, polymorphic information content (PIC), and observed and expected heterozygosity all assessed. Overall, sequence-based allelic variants of the repeat region were observed in 20 out of 22 different STR loci commonly used in forensic DNA genotyping, with the highest number of sequence variation observed at locus D12S391. The highest increase in allelic diversity and in PIC through sequence-based genotyping was observed at loci D3S1358 and D8S1179. Such detailed sequence analysis, as the one performed in the present study, is important to help understand the diversity of sequence-based STR alleles across different populations and to demonstrate how such allelic variation can improve statistics used for forensic casework.
Asunto(s)
Dermatoglifia del ADN , Genética de Población , Secuenciación de Nucleótidos de Alto Rendimiento , Repeticiones de Microsatélite , Grupos Raciales/genética , Electroforesis Capilar , Femenino , Frecuencia de los Genes , Genotipo , Heterocigoto , Humanos , Masculino , Polimorfismo Genético , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
The use of environmental trace material to aid criminal investigations is an ongoing field of research within forensic science. The application of environmental material thus far has focused upon a variety of different objectives relevant to forensic biology, including sample provenance (also referred to as sample attribution). The capability to predict the provenance or origin of an environmental DNA sample would be an advantageous addition to the suite of investigative tools currently available. A metabarcoding approach is often used to predict sample provenance, through the extraction and comparison of the DNA signatures found within different environmental materials, such as the bacteria within soil or fungi within dust. Such approaches are combined with bioinformatics workflows and statistical modelling, often as part of large-scale study, with less emphasis on the investigation of the adaptation of these methods to a smaller scale method for forensic use. The present work was investigating a small-scale approach as an adaptation of a larger metabarcoding study to develop a model for global sample provenance using fungal DNA signatures collected from dust swabs. This adaptation was to facilitate a standardized method for consistent, reproducible sample treatment, including bioinformatics processing and final application of resulting data to the available prediction model. To investigate this small-scale method, 76 DNA samples were treated as anonymous test samples and analyzed using the standardized process to demonstrate and evaluate processing and customized sequence data analysis. This testing included samples originating from countries previously used to train the model, samples artificially mixed to represent multiple or mixed countries, as well as outgroup samples. Positive controls were also developed to monitor laboratory processing and bioinformatics analysis. Through this evaluation we were able to demonstrate that the samples could be processed and analyzed in a consistent manner, facilitated by a relatively user-friendly bioinformatic pipeline for sequence data analysis. Such investigation into standardized analyses and application of metabarcoding data is of key importance for the future use of applied microbiology in forensic science.
Asunto(s)
Código de Barras del ADN Taxonómico , ADN de Hongos/química , Hongos , Suelo , Demografía , Ciencias Forenses , Humanos , Valores de ReferenciaRESUMEN
DNA sequencing technologies continue to improve, and there has been a corresponding expansion of DNA-based applications in the forensic sciences. DNA recovered from dust and environmental debris can be used to identify the organisms associated with these sample types, including bacteria, plants, fungi, and insects. Such results can then be leveraged to discern sample origin or geolocation and investigate individual identification. Here, we take a critical look at the current DNA-based technologies using microbiome and environmental sample sources that are focused on the generation of some investigative tools for use in forensic science. We discuss the pitfalls and contentions associated with the use of these techniques and highlight some of the future research required to expand the utility of these methods in the forensic sciences.
Asunto(s)
Bacterias/aislamiento & purificación , ADN Ambiental/análisis , Ciencias Forenses , Hongos/aislamiento & purificación , Plantas , Código de Barras del ADN Taxonómico , Ciencias Forenses/instrumentación , Ciencias Forenses/métodos , Humanos , Microbiota , Microbiología del SueloRESUMEN
Assigning the gender of a DNA contributor in forensic analysis is typically achieved using the amelogenin test. Occasionally, this test produces false-positive results due to deletions occurring on the Y chromosome. Here, a four-marker "YFlag" method is presented to infer gender using single-base extension primers to flag the presence (or absence) of Y-chromosome DNA within a sample to supplement forensic STR profiling. This method offers built-in redundancy, with a single marker being sufficient to detect the presence of male DNA. In a study using 30 male and 30 female individuals, detection of male DNA was achieved with c. 0.03 ng of male DNA. All four markers were present in male/female mixture samples despite the presence of excessive female DNA. In summary, the YFlag system offers a method that is reproducible, specific, and sensitive, making it suitable for forensic use to detect male DNA.
Asunto(s)
Cromosomas Humanos Y/genética , Cartilla de ADN , Análisis para Determinación del Sexo/métodos , Proteínas de Ciclo Celular/genética , Dermatoglifia del ADN , Femenino , Genes sry/genética , Marcadores Genéticos , Humanos , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Polimorfismo de Nucleótido Simple , Proteínas/genética , Reproducibilidad de los ResultadosRESUMEN
The ability to predict externally visible characteristics (EVCs) from DNA has appeal for use in forensic science, particularly where a forensic database match is not made and an eye witness account is unavailable. This technology has yet to be implemented in casework in New Zealand. The broad cultural diversity and likely population stratification within New Zealand dictates that any EVC predictions made using anonymous DNA must perform accurately in the absence of knowledge of the donor's ancestral background. Here we construct classification tree models with SNPs of known association with eye colour phenotypes in three categories, blue vs. non-blue, brown vs. non-brown and intermediate vs. non-intermediate. A set of nineteen SNPs from ten different known or suspected pigmentation genes were selected from the literature. A training dataset of 101 unrelated individuals from the New Zealand population and representing different ancestral backgrounds were used. We constructed four alternate models capable of predicting eye colour from the DNA genotypes of SNPs located within the HERC2, OCA2, TYR and SLC24A4 genes using probability calculation and classification trees. The final model selected for eye colour prediction exhibited high levels of accuracy for both blue (89%) and brown eye colour (94%). Models were further assessed with a test set of 25 'blind' samples where phenotype was unknown, with blue and brown eye colour predicted correctly where model thresholds were met. Classification trees offer an aesthetically simple and comprehendible model to predict blue and brown eye colour.