Abstract 1413: Automation of a barcoded ctDNA sequencing assay based on hybrid capture on two robotic pipetting platforms enables processing of large numbers of liquid biopsy samples
Authors: Lange Maria , Siemann Sandra , Woestmann Corinna , McNamara Sylvie , Feldkamp Mirjam , Blueher Anja , Hinzmann Bernd , Rosenthal Andre 
Published: August 2018

Abstract
Introduction: Today's ctDNA sequencing assays are either lab-developed tests (LDTs) and offered as centralized service by CLIA labs, or distributed kits (Qiagen, Roche Avenio ctDNA kit) that require manual processing of samples. The AVENIO ctDNA assays (RUO, Roche Molecular Solutions, Pleasanton CA, USA) are based on the hybrid capture technology that utilizes molecular barcoding and detects four mutation classes (SNV, IDels, CNVs, Fusions). The current manual presequencing workflow requires three full working days. With the recent progress in sequencing of longitudinal liquid biopsy samples there is a clear need for automation of the workflow. Here, we present two automated workflows on the Perkin Elmer NGS Sciclone and a modified Hamilton Star.
Methods: Batches of 24 samples of 50ng genomic DNA and 24 samples of 50ng DNA from Horizon cell line blends (HD70, HD731, HD733, HD734) were tested in microtiter plates using manual and automated workflows for comparison. AVENIO cfDNA extraction kit and MagnaPure 96 (MP96) were used for manual and automated extractions of 2 mL of plasma. The AVENIO ctDNA assay was used for manual processing of library prep and target enrichment according to the published instructions for use. Two pipetting robots were used for an automated version of the AVENIO ctDNA workflow. The Perkin Elmer Sciclone has a 96 pipetting head and can process 96 samples in 2.5 days. The modified Hamilton Star has a 8 pipetting head and can process 24 samples in 3 days. After library prep and target enrichment, pools of 12 samples were run on the NextSeq. Sequencing QC parameters were analyzed with focus on on-target rate, duplication rate, deduped depth and insert sizes.
Results: An automated workflow for the commercially available AVENIO ctDNA assay was successfully established on both the Perkin Elmer Sciclone and modified Hamilton Star. The main sequencing QC parameters were very similar between Sciclone and modified Star. There was no difference in the somatic calls for the HD cell lines from 5% AF (HD730, 731) down to 2% AF (HD733) and 1.3% AF (HD734) for the manual workflow compared to the two automated workflows. Finally, two sets of 24x 2mL plasma samples from 24 patients with metastatic CRC were processed using (a) the manual cfDNA extraction and AVENIO ctDNA workflow, and (b) automated cfDNA extraction (MP96) and automated workflow on the Sciclone. No major difference was observed between the sequencing QC parameters.
Conclusions: We have successfully automated the AVENIO ctDNA workflow on both the Perkin Elmer Sciclone and a modified Hamilton Star. Performance characteristics are very similar on both platforms with respect to on-target and duplication rates, deduped depth and insert sizes. The Sciclone is a widely available pipetting robotic liquid handler and can support processing between 8-192 samples per week with one operator.
Citation Format: Maria Lange, Sandra Siemann, Corinna Woestmann, Sylvie McNamara, Mirjam Feldkamp, Anja Blueher, Bernd Hinzmann, Andre Rosenthal. Automation of a barcoded ctDNA sequencing assay based on hybrid capture on two robotic pipetting platforms enables processing of large numbers of liquid biopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1413.