Evaluation of microhaplotypes in forensic kinship analysis from a Swedish population perspective

Adam Staadig,Andreas Tillmar

doi:10.1007/s00414-021-02509-y

Abstract

The development of massively parallel sequencing (MPS) technology has enabled the discovery of several new types of forensic markers where microhaplotypes are one of these promising novel genetic markers. Microhaplotypes are, commonly, less than 300 nucleotides in length and consist of two or more closely linked single-nucleotide polymorphisms (SNPs). In this study, we have examined a custom-made QIAseq Microhaplotype panel (Qiagen), including 45 different microhaplotype loci. DNA libraries were prepared according to the GeneRead DNAseq Targeted Panels V2 library preparation workflow (Qiagen) and sequenced on a MiSeq FGx instrument (Verogen). We evaluated the performance of the panel based on 75 samples of Swedish origin and haplotype frequencies were established. We performed sensitivity studies and could detect haplotypes at input amounts down to 0.8 ng. We also studied mixture samples with two contributors for which haplotypes, for the minor contributor, were detectable down to the level of 1:100. Furthermore, we executed kinship simulations to evaluate the usefulness of this panel in kinship analysis. The results showed that both paternity and full sibling cases can clearly be solved. When simulating a half sibling versus unrelated case scenario, there were, however, some overlap of the likelihood ratio distributions potentially resulting in inconclusiveness. To conclude, the results of this initial study are promising for further implementation of this microhaplotype assay into the forensic field, although we noticed some primer design issues that could be optimized, which possibly would increase the power of the assay.

Highlights

The forensic field is in a continuous need of improvements due to the various and complex issues in forensic investigations, in addition with its often-intricate sample material and quality
Standard short tandem repeat (STR) analysis has limitations when working with highly degraded samples due to their relatively long fragment length
In human identification cases, the sample material can often be degraded and fragmented, wherefore the relatively long STR markers can result in incomplete DNA typing and the shorter microhaplotypes are believed to be a more appropriate marker

Summary

Introduction

The forensic field is in a continuous need of improvements due to the various and complex issues in forensic investigations, in addition with its often-intricate sample material and quality. Standard short tandem repeat (STR) analysis has limitations when working with highly degraded samples due to their relatively long fragment length. It is known that the mutation rates among SNPs [5] are much lower than for STR markers [6, 7]. All these features make microhaplotypes a suitable marker of choice in missing person identification where the reference individual is a close relative. In human identification cases, the sample material can often be degraded and fragmented, wherefore the relatively long STR markers can result in incomplete DNA typing and the shorter microhaplotypes are believed to be a more appropriate marker. Microhaplotypes could be a useful tool for predicting the biogeographic ancestry of an individual, which could be an important investigative lead in criminal investigations [2]

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Conclusion