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Validation between manual and automated NGS library prep

In forensic practice, library preparation for sequencing has been identified as a significant bottleneck to broader adoption. Next Generation Sequencing (NGS) library preparation is usually a multistep process and requires costly reagents and substantial hands-on operations. To ensure a high degree of robustness and reproducibility of results, standardized sample preparation approaches and quality control measures are critical. Automation solutions can perform complex protocols with high reproducibility, leading to reduced error rates, while reducing risks associated with contamination, human interaction, reagents and samples. Overall, cost per sample can also be lowered by decreasing the hands-on time and labor associated with the workflow.

To implement automation in the process of NGS, DNA Labs International in collaboration with Verogen and Aurora validated ForenSeq kits on the VERSA 1100 NGS instrument (Figure 1). The validation was performed for the ForenSeqTM Kintelligence Kit, the ForenSeqTM mtDNA Whole Genome Kit, and the ForenSeqTM Signature Prep Kit (DNA Primer Mix B or DPMB). This application note compared libraries generated using manual and automated workflow and sequenced on the MiSeq FGx sequencing system.

Two replicates of libraries were processed manually while two replicates of libraries were processed using the Aurora’s VERSA 1100. All samples processed on the Aurora’s VERSA 1100 generated a similar number of reads to those that were processed manually. The total sample reads for each sample across manual and automated set-ups were plotted against DNA input ranging from 25 pg to 1000 pg. For detailed call rate results, read the full validation . All samples met the upload criteria for GEDmatch PROTM. Moreover, sensitivity studies were conducted to evaluate the performance of the system over a range of DNA quantities to inform profile result expectations and identify interpretation limitations. Both manual and automated systems produced comparable results for a dynamic range of sample inputs (8 pg to 4000 pg).

The sensitivity, reliability, repeatability, concordance, ability to operate with minimal risk of contamination, reproducibility, and accuracy were demonstrated using an automated workflow. These internal validation studies support the use of Aurora’s VERSA 1100 when used with Verogen products for sequencing forensic samples. The validation showed the ability of Aurora’s VERSA 1100 to efficiently automate NGS workflows in alignment with forensic standards. For full results and details please read the Validation Report.