Abstract 3141: Pro-Seq: A novel method to improve sequencing accuracy for liquid biopsy of ctDNA from healthy individuals and cancer patients
Authors: Marziali Andre 
Published: July 2016

A significant barrier to widespread clinical deployment of sensitive circulating tumor DNA (ctDNA) assays (liquid biopsy) is the high assay cost compared to potential reimbursement. Assay cost is currently dominated by the large amount of DNA sequencing required to achieve coverage of a broad gene panel, and by the high read depth required for high clinical sensitivity. Attaching unique molecular barcodes to ctDNA fragments for the purpose of error reduction further increases sequencing requirements, making liquid biopsy commercialization in many clinical applications impractical.

We present a novel library construction process for NGS sequencing that increases the accuracy of the combined library construction and sequencing process by an order of magnitude. Named Proximity-Sequencing (Pro-Seq), the method duplicates the sequence information in each original DNA strand prior to the bulk of library construction in such a way as to provide redundant, linked templates to the sequencer. The redundant templates remain linked through the library construction process, allowing detection of PCR errors as sequence disagreement between the two strands. The linked templates are amplified in a single sequencing reaction, such that base quality and incorporation information can be used to determine which bases of the sequence were corrupted during PCR amplification steps. Since both strands are amplified as part of the same sequencing read, sequencing accuracy is improved without requiring use of additional reads on the sequencer.

A key element of this process is a novel linked-linear amplification in which DNA primers linked by a short molecule amplify a single strand in the same sense, ensuring twin copies in the same sense that remain physically linked. The method is entirely based on novel reagents and can be implemented without additional instrumentation beyond standard NGS equipment.

The method is expected to have significant utility in any applications that require detection of rare sequence variants, including analysis of cell free DNA for liquid biopsy applications. We demonstrate the ability to achieve sequence accuracy similar to barcoded sequencing methods, without the additional sequencing burden required by such methods.

We present the method using an Illumina platform, and present data from sequencing of bacterial and human cell free DNA that demonstrate error rates that are improved by an order of magnitude over the current state of the art NGS chemistries. The addition of Pro-Seq library construction to NGS assays enables lower cost, high sensitivity, and high specificity liquid biopsy tests to be developed, enabling commercialization in applications with limited reimbursement, potentially including early cancer detection.

Citation Format: Andre Marziali. Pro-Seq: A novel method to improve sequencing accuracy for liquid biopsy of ctDNA from healthy individuals and cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3141.