Massively Parallel Sequencing (MPS) technology can be used to supplement traditional DNA profiling in various ways. In this work, we describe the experience of in-house implementation of two MPS-based methods at the Swedish National Forensic Centre. First, the ForenSeq Imagen kit, which is applied to generate a prediction of phenotype characteristics (hair-, eye- and skin colour) and an assessment of biogeographical ancestry for the donor of a trace sample. The results are reported as a forensic intelligence lead in cases where no match has been obtained with traditional STR-based DNA profiling. Secondly, the ForenSeq Mainstay SE kit, which is used to sequence STR markers and can be applied to obtain improved results for degraded samples and complex mixtures.

For both methods, the workflow begins with a request from case investigators. If a suitable DNA trace sample is available in the case, the analysis starts by preparing a DNA sequencing library from the DNA extract using reagents specific to the selected kits. Sequencing is performed using a MiSeq FGx instrument followed by bioinformatic analysis. The HIrisPlex-S model is used to predict phenotype characteristics. For ancestry, three different software tools (Snipper, GenoGeographer and STRUCTURE) are used to analyse the data in addition to the analysis tool provided by the kit manufacturer. The prediction results are interpreted together and combined as a final output. When the Mainstay kit is used, the obtained sequence-based STR profiles may be used for national database searches and/or matching to individuals analogous to results gained from capillary electrophoresis-based STR profiling.

The ForenSeq Imagen kit has been in use for close to two years and the experience from this first period of application in casework is presented. The Mainstay kit has recently been operationalized. For each method, we describe the validation approach as well as practical challenges encountered in application of new methods into casework, including harmonizing the interpretation and reporting of results. We will also present analytical success rates for various sample types and suggest selection criteria for identifying suitable samples for the methods.

Authors

• Karin Westerberg (National Forensic Centre, Swedish Police Authority, Sweden)
• Arvid Hedén Gynnå (National Forensic Centre, Swedish Police Authority, Sweden)
• Siri Aili Fagerholm (National Forensic Centre, Swedish Police Authority, Sweden)
• Nelly Gyllö Lind (National Forensic Centre, Swedish Police Authority, Sweden)
• Ricky Ansell (National Forensic Centre, Swedish Police Authority and Department of Physics, Chemistry & Biology, Linköping University, Sweden)
• Henric Bergman (National Forensic Centre, Swedish Police Authority, Sweden)
• Ronny Hedell (National Forensic Centre, Swedish Police Authority, Sweden)
• Maja Sidstedt (National Forensic Centre, Swedish Police Authority, Sweden)