Challenge Met! Forensic Genetics Laboratory Fully Converted to MPS

The use of massive parallel sequencing (MPS) is being incorporated quickly into forensic genetics. Although it represents a much more complex technology than capillary electrophoresis (CE), its advantages are increasingly evident.

Here we present our experience of transforming a CE-based into an MPS-based forensic laboratory, already working under 17025 standards, and how we have applied this workflow to the identification of challenging historical samples. We presented our accreditation work to achieve ISO/IEC 17025 for ForenSeq® DNA Signature Prep Kit and ForenSeq®MainstAY Kit sequencing on a MiSeqFGx® platform at the ISFG conferences 2019 and 2022, respectively.

Since then, we have established a unique workflow for processing reference samples based on the ForenSeq®MainstAY kit, typing 27 aSTRs and 25 Y-STRs in one single reaction with a regular DNA input of 0.25ng, with accuracy, sensitivity and call rate values > 99%. This allows the laboratory to be more efficient.

As a laboratory working in missing persons projects form the Spanish Civil War (1936-1939) we have already studied more than 120 ~90 years old bone samples applying MPS, obtaining full profiles (27 aSTRs and 25 Y STRs) from degraded and low concentrated samples (quant values in range 10^-2 to 10^-3 ng/µL) with different degradation indexes, when working with ForenSeq® MainstAY kit. These studies have enabled us to identify several missing persons, obtaining quite high LR values (up to 49500 in grandfather-grandson cases) in the kinship studies based on autosomal STRs, and in many cases, also supported by Y-STRs results. We present a case study of eight unidentified remains found in a grave (killed in 1939) where we were able to identify 6 individuals based on aSTRs and Y STRs. Samples presented DI from 4 to 10 and quantification values in the above-mentioned range. Kinship calculations were carried out with the DVI FAMILIAS 3 module for aSTRs and based on YHRD database for Y-STRs.

We have also applied the ForenSeq® DNA Signature Prep Kit to the study of historical remains from the XVI century obtaining not only very good STR profiles but also identity, phenotypic and biogeographical ancestry related SNPs, that allowed us to do estimations.

Because many of our samples are very old bones, our laboratory also performs mitochondrial control region analysis. We were able to obtain complete mitochondrial control region (HVI, HVII and HVIII) sequences starting from bone extracted DNA with quantification values in the range of 10^-4 ng/µL. This sample didn’t perform with the STR kits, but we got well over 100.000 reads/sample when sequencing the CR of mtDNA with the ForenSeq® mtDNA CR kit.

As a conclusion, we have successfully implemented MPS in our laboratory. In our experience, MPS allows us to be more efficient in our routine work with reference samples and is invaluable when dealing with highly degraded bone samples. Next Generation sequencing is no longer a tool for exceptional cases.