For the past 20 years, exploiting length-based differences in short tandem repeats (STRs) through capillary electrophoresis (CE) has been a primary method relied upon by forensic DNA testing laboratories. However, massively parallel sequencing (MPS) is emerging within the forensic community as a potential DNA typing technology. In contrast to CE, STR typing by MPS, yields both size- and sequence-based information. Integration of MPS into forensic DNA workflows provides value by enhancing the analysis of degraded samples; reducing the cost of materials and labor time through the use of large multiplexes containing expanded DNA marker sets; the separation of mixtures; and the capability of using phenotypic markers to further characterize through physical characteristics.
Submitted by: Liz Montano, Battelle
To efficiently integrate MPS into an accredited forensic DNA laboratory, special considerations arise, as DNA typing by MPS includes additional laboratory steps and many of the current MPS methods are designed for research laboratories with little experience with forensic practices.
In this study, Battelle scientists collaborated with members of the CODIS laboratory at the Ohio Bureau of Criminal Investigation (OBCI) to optimize the Promega PowerSeq Auto/Y System with modifications designed to improve sample processing in a forensic DNA laboratory. The system includes Promega’s PowerSeq Auto/Y prototype MPS amplification kit, Illumina’s TruSeq® DNA PCR-Free library prep kits, the Illumina MiSeq sequencer in Research-Use-Only mode with MiSeq v2 300-cycle sequencing kits. Battelle and OBCI identified the following modifications for integration of the PowerSeq Auto/Y System into a forensics laboratory: 1) Remove non-essential quality control checks 2) Replace purification columns with bead purification procedures 3) Automate the laboratory workflow 4) Remove End-Repair steps 5) Remove quantification steps of amplified product. Experiments assessed streamlining some quality control steps, adjusting sample purification procedures, incorporating sample-processing automation, and reducing the amount of hands-on manipulation of amplified products. To compare data quality from each adjustment, identical samples were processed side-by-side using both the original and the optimized procedures. Amplification products were assessed using Agilent Bioanalyzer 2100 electrophoretic traces, and sequencing data was processed using Battelle’s ExactID® software.
Experimental testing showed that all modifications, apart from removing End Repair, simplified the MPS forensic sample processing and had no negative impact on data quality. Research suggests the PowerSeq amplification primers are likely to be un-phosphorylated, thus requiring these amplification products still undergo the 5` phosphorylation reaction that takes place during End Repair steps to allow for efficient library preparation. Results from all other laboratory evaluations demonstrated that such methodology enhancements effectively streamlined sample processing while maintaining data quality levels.
The original laboratory method involved 12 separate steps, 3 new pieces of instrumentation, almost 3 days of hands-on time for the analyst, increased material costs, and steps unfavorable with an accredited, case-work forensic laboratory. Battelle and OBCI evaluated improvements in the workflow that reduce the chances of sample contamination, the chances of human error (sample switching), hands-on times with automation, sample turnaround time, and the costs of both instrumentation and reagents. Overall, this study shows that the Promega PowerSeq Auto/Y system can be optimized to be used in a forensic setting, and that with optimizations, MPS procedures can be integrated into a forensic DNA laboratory. In future studies, Battelle and OBCI are further developing this MPS workflow for implementation into a forensic laboratory.
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