Considerations for the Implementation of Massively Parallel Sequencing into Routine Kinship Analysis

Background: Investigating the way in which individuals are genetically related has been a long-standing application of forensic DNA typing. Whilst capillary electrophoresis (CE)-based STR analysis is likely to provide sufficient data to resolve regularly encountered paternity cases, its power to adequately resolve more distant or complex relationships can be limited. Massively parallel sequencing (MPS) has become a popular alternative method to CE for analysing genetic markers for forensic applications, including kinship analysis. Data workflows used in kinship testing are well-characterised for CE-based methodologies but are much less established for MPS. When incorporating this technology into routine relationship casework, modifications to existing procedures will be required to ensure that the full power of MPS can be utilised whilst maintaining the authenticity of results. Methods: Empirical data generated with MPS for forensically relevant STRs and SNPs and real-world case experience have been used to determine the necessary workflow adaptations. Results: The four considerations highlighted in this work revolve around the distinctive properties of sequence-based data and the need to adapt CE-based data analysis workflows to ensure compatibility with existing kinship software. These considerations can be summarised as the need for a suitable sequence-based allele nomenclature; methods to account for mutational events; appropriate population databases; and procedures for dealing with rare allele frequencies. Additionally, a practical outline of the statistical adjustments required to account for genetic linkage between loci, within the expanded marker sets associated with MPS, has been presented. Conclusions: This article provides a framework for laboratories wishing to implement MPS into routine kinship analysis, with guidance on aspects of the data analysis and statistical interpretation processes.