Microhaplotype loci are a powerful new type of forensic marker

Modern sequencing technology makes it possible to genotype polymorphisms with high throughput and high multiplexing. We have searched for and identified many loci with 2 or more SNPs within the expanse of a 200 bp single sequence run and show that when linkage disequilibrium is not complete these loci have multiple alleles detected as phase-known haplotypes. These microhaplotype loci (microhaps) are a powerful tool for individual identification, ancestry inference, and determining family/clan relationships.     

Introduction of the python script MHinNGS for analysis of microhaplotypes

MHinNGS is a Python application developed for analysis of microhaplotypes (MHs) in single-end sequencing data. MHinNGS analyses reads in standard formats and store each sequence into bins, one bin for each MH as defined by the two flanking sequences. MHinNGS requires a reference genome and a configuration file with information about each locus. Four mandatory and 15 optional criteria defined in the configuration file allow detailed locus-specific analyses of the MH loci. The program 1) removes noise, 2) identify and name alleles, 3) test the genotypes, and 4) test unique sequences not identified as noise or alleles. MHinNGS produces a result file, where every unique sequence that passed the noise filter is presented with MH allele, read depth, warning flags based on the genotyping criteria, sequence, heterozygote balance, and MH name. Furthermore, variation in other parts of the fragment that is not defined as SNPs in the MH, linked variants, or rare SNPs are listed in a separate column of the result file.     

Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers

When using a standard battery of STRs for relationship testing a small proportion of analyses can give ambiguous results – where the claimed relationship cannot be confirmed by a high enough paternity index or excluded with fully incompatible genotypes. The majority of such cases arise from unknowingly testing a brother of the true father and observing only a small number of exclusions that can each be interpreted as one- or two-step mutations. Although adding extra STRs might resolve a proportion of cases, there are few properly validated extra STRs available, while the commonly added hypervariable SE33 locus is four times more mutable than average, increasing the risk of ambiguous results. We have found SNPs in large multiplexes are much more informative for both low initial probabilities or ambiguous exclusions and at the same time provide a more reliable genotyping approach for the highly degraded DNA encountered in many identification cases. Eight relationship cases are outlined where the addition of SNP data resolved analyses that had remained ambiguous even with extended STR typing. In addition we have made simulations to ascertain the frequency of failing to obtain exclusions or conclusive probabilities of paternity with different marker sets when a brother of the true father is tested. Results indicate that SNPs are statistically more efficient than STRs in resolving cases that distinguish first-degree relatives in deficient pedigrees.     

SNP分型方法及在法医学等领域的应用

随着单倍型图的产生,SNP越来越受到关注。不仅仅在Y染色体和线粒体,常染色体和X染色体上的SNPs的应用潜能也将被发现。SNPs具有比较低的突变率和适合于降解DNA分析的特点,在法医学领域也受到关注。本文综合介绍了SNPs和X-SNPs的一般特性、分型方法及其在法医学的应用。     

Development of Forensic Assay Signatures for Ebolaviruses

Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan‐MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 “unknown” ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high‐resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.     

Population data for 94 identity SNPs in four U.S. population groups

The U.S. National Institute of Standards and Technology (NIST) sequenced 1036 human DNA samples from four United States population groups (African American, Asian, Hispanic, and Caucasian) using the ForenSeq DNA Signature Prep Kit with Primer Mix B (DPMB) on a MiSeq FGx instrument. In addition to STR markers, DPMB includes amplification primers for single nucleotide polymorphisms (SNPs) used for individual identification (iiSNPs, n = 94), ancestry inference (aiSNPs, n = 56), and phenotype prediction (piSNPs, n = 22). Resulting sequencing coverage information was interpreted for the 94 iiSNP markers. Here we present performance characteristics of the ForenSeq DNA Signature Prep Kit in the population studied.     

Make it "SNPPY" - Updates to SRM 2391d: PCR-Based DNA Profiling Standard

Standard Reference Material (SRM) 2391d: PCR-Based DNA Profiling Standard was released to the forensic community in 2019. Next Generation Sequencing (NGS) was used as the primary method of certification, where certified values were assigned when a high coverage sequence string was available for a marker. Using NGS to assign values has allowed for additional marker sets beyond short tandem repeat (STR) loci, including single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) whole genome sequences, to be included in the Certificate of Analysis (COA). Since the 2019 release, several commercial NGS panels have become available including the Verogen ForenSeq mtDNA Control Region, mtDNA Whole Genome, MainstAY, and Kintelligence Kits. In addition, three community Ion AmpliSeq panels from Thermo Fisher (MH-74 Plex, VISAGE, and Y-SNP) are now available. While the mtDNA whole genome sequence for the components are already included and no new STR markers are introduced by MainstAY, the other recently released panels allow for the inclusion of > 11,000 additional SNPs (e.g., identity, ancestry, phenotype, kinship, and X- and Y-SNPs) and 74 microhaplotypes to the COA for SRM 2391d in an update completed by fall of 2022.     

Analysis of genetic polymorphisms associated with the presence of freckles for phenotypic prediction

The prediction of externally visible characteristics (EVCs) is a commonly used practice by the forensic sciences as an important resource in the investigation of criminal cases in which the identity of perpetrators or victims is unknown or even to recognize decomposed cadavers. With this purpose, genetic markers associated with pigmentation traits have been widely studied by forensic scientists and, nowadays, it is possible to predict phenotypic characteristics such as hair, eyes and skin colour, as well as the presence of skin freckles by analysing single nucleotide polymorphisms (SNPs). In this study, we analysed the association of six SNPs located in pigmentation genes to the presence of freckles in individuals from the Brazilian population for forensic DNA phenotyping. The study was based within the context of a larger project on a population sample of 534 adult Brazilians of both sexes and different skin colours. DNA was extracted from peripheral blood and genotyped using the TaqMan® OpenArray® Real-Time PCR System (ThermoFischer Scientific) technique. Statistical analyses were carried out with the R software (version 4.0.2). As for the results obtained, three SNPs were shown to be statistically associated to the freckling, rs12203592, rs1800404 and rs222847, with CT, AG and AA genotypes being the main contributors, respectively. Variables such as sex of the individuals and skin colour were found to also contribute to the manifestation of this pigmentation trait. Further statistical analyses will be carried out to evaluate the possibility of using the SNPs in this study for phenotyping prediction of the Brazilian population, improving existing DNA phenotyping models in forensic sciences.