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Assessment of massively parallel sequencing platform MiSeqTM FGx and ForenSeqTM DNA Signature Prep Kit prior to introduction into routine forensic casework

In this era of fast evolving new technologies, massively parallel sequencing (MPS) powered the analysis of vast amount of DNA markers in a single reaction. However, increased number of markers and gain of sequence information make analysis and interpretation of results more challenging. In order to assess the applicability of MiSeqTM FGx (Illumina®) system and ForenSeqTM DNA Signature Prep Kit (Illumina®) in our forensic laboratory workflow, the following experiments were conducted. Repeatability and reproducibility: triplicates of positive control 2800M and two reference samples in two independent runs. Sensitivity: triplicates of dilution series of 2800M (ranging from 1 ng to 0.063 ng). Concordance: comparison of STR profiles from capillary electrophoresis (CE) to MPS results for 16 reference samples. Libraries were prepared according to manufacturer’s instructions, using ForenSeqTM DNA Signature Prep Kit with DNA Primer Mix B, and pooled for sequencing on MiSeqTM FGx instrument. Subsequent data analysis was performed by ForenSeqTM UAS software (Illumina®, v.1.3). Overall, run metrics (cluster density, clusters passing filter) were within optimal range. Results showed fully reproducible STR profiles for all replicates of 2800M control and two reference samples. At recommended DNA input of 1 ng, all STR loci were typable for all replicates of 2800M control. Intralocus imbalance and elevated stutters became more prominent with lower DNA input. At 0.063 ng some loci drop-outs were observed, but the majority were still typable, thus proving high sensitivity of the kit. Concordant STR profiles between CE and MPS were obtained for all 16 reference samples. Also, phenotypic and ancestry information were successfully obtained in all instances. This assessment is an ongoing process, leading towards internal validation, with the goal of implementing MiSeqTM FGx instrument and ForenSeqTM DNA Signature Prep Kit into forensic casework routine.

massively parallel sequencing, MiSeq FGx, ForenSeq

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