Are microhaplotypes derived from the 1000 Genomes Project reliable for forensic purposes?

Microhaplotypes (MHs) have emerged as an important genetic marker in forensic genetics. However, most studies have overlooked the potential for phasing errors within microhaplotypes based on the 1000 Genome Project (1kGP), which may impact the evaluation of various forensic parameters and lead to misleading results. In this study, we constructed a dense and extensive set of MHs across the human genome, using the expanded 1000 Genomes Project data aligned to GRCh38 reference genome. We applied three different SNP minor allele frequency (MAF) thresholds (0, 0.01, and 0.05) to evaluate the reliability of these markers. Utilizing pedigree data from 18 populations, which included a total of 602 trios, we scanned for and confirmed suspected phasing error events at these MH loci. We also sequenced 50 MHs for one trio of the Southern Han Chinese (CHS) population to further investigate these discrepancies. The results revealed the presence of phasing errors in MHs from 1kGP when analyzed using targeted enrichment and next-generation sequencing. The probability of suspected phasing error events was strongly and positively correlated with the effective number of alleles (Ae) and informativeness (In) of the markers. Additionally, these mismatch probabilities varied significantly across different continental populations. Additionally, when selecting loci, applying MAF filtering and avoiding regions such as the MHC can reduce the occurrence of such events to some extent. Based on these findings, we suggest that relying solely on sequencing data of the 1kGP for forensic purpose may be risky. A thorough investigation of the true forensic parameters of MHs is essential to ensure their reliability in forensic applications.