Microhaplotypes in forensic genetics: From exploration to application in degraded DNA specimens

Microhaplotypes have emerged as powerful forensic markers over the past decade. This paper sets out the development of a MPS panel of microhaps and its potential for application to identification, analysis of degraded DNA, ancestry inference, and identification of close biological relationships. To make it more effective when dealing with fragmented DNA, the MPS assay was designed to ensure a reduced amplicon size of less than 140 bp. After MPS assay validation, a panel of 76 microhaps, comprised of 299 different SNPs and spread across the autosomal human genome, was established. A total of 102 Italian individuals were analyzed to estimate the genotype and haplotype frequencies. The effective number of alleles at each locus (A_e) for the Italian population ranges from 1.926 to 6.187, with 59 MHs that have values greater than 3.0. The matching probability (PI) ranges from 0.055 to 0.345 and the cumulative PI value is 11.763E-66. Complete and reliable profiles were obtained with as little as 0.05 ng. The MHs panel was then validated on real forensic specimens chosen on the basis of their DNA content and degradation level. The majority of the casework samples analyzed showed complete or nearly complete MH profiles even in degraded samples. To assess the informative power of MH profiles in forensic casework, probabilistic genotyping on partial MH profiles has been used. The resulting likelihood ratio values range from 7.84E+09 to 2.70E+34, thus defining an extremely strong support for the hypothesis that the genetic profile in a casework sample comes from the reference sample. Pairwise kinship simulations using allele frequencies from Italian population samples showed that full- and half-sibling relationships can be readily distinguished from unrelated individuals. For evaluation of the 76 MH panel's utility for ancestry informativeness, PCA and STRUCTURE analyses are also presented comparing the newly collected sample from Ancona Italy with the 26 populations of the 1000 Genomes Project. The results of the analysis confirmed the effectiveness of these short microhaplotypes in typing, with high sensitivity, samples with highly degraded DNA typically encountered in forensic cases.