For abroad application of liquid biopsy in routine clinical settings, it is essential to use a high performance target-enrichment technique that able to detect all major classes of mutations including single nucleotide variants (SNVs), insertion/deletions (InDels), copy number alternations (CNVs) and rearrangements with affordable costs. The regular enrichment strategies employ unique molecular index (UMI) to suppress sequencing error are limited by several factors for their widespread adoption in clinical application. As an instance for hybridization based methods, Cancer Personalized Profiling by deep Sequencing (CAPP-SEQ) is expensive due to low on target rate. Oncomine, as an example for multiple PCR based technique, is unable to detect DNA rearrangements. Herein, we propose a novel target-enrichment strategy based on the innovated sequencing platform BGISEQ-500. The probes used here are blocked by ddNTPs at 3' end which suit for pyrophosphorolysis-activated polymerization. Modified probes in 3' end consist of complementary sequences targeting SNVs, InDel's hotspots, frequently rearranged intron regions, and a sequencing platform-specific tail in 5' end. In a multi-cycle hybridization and copy procedure with prepared library for enrichment, probes work in two roles: hybridization baits and primers for successively extension; while in Post-PCR, they act as universal PCR sequence to produce sequencing library. Since each molecule containing target locus as well as forward UMI adapters will be captured and copied, we could distinguish the origin of each reads by pull-down probes among huge sequencing data, and select probes with high performance to construct clinical NGS panels. Meanwhile, intermediate and final products from the capture process will be gathered for quality control to evaluate the annealing efficiency and on-target ratio. QPCR primers for EGFR and KRAS are included in the panel to represent areas with normal/low GC content. The collected data were useful to monitor the loss of molecules during hybridization and provide key information for further improvement of the panel. For a customized sequencing panel of non-small-cell lung carcinoma (NSCLC) with sequencing regions about 30k, the sequencing results showed overall very high percentage of reads on target (>95%) and very high data utilization rate (>75%). With 40ng DNA used as input for library generation and 4 Gbyte data size, we can achieve 8000X de-duplicate sequence depth, of which 5000X is error corrected depth, thereby ensure the stable detection of mutation with frequency as low as 0.1%. We believe this novel capture and analysis technique is also applicable to other capture-based sequencing product and will achieve similar performance. Cancer patients, especially NSCLC will benefit from this new sequencing technique.

Citation Format: Jilong Liu, Yan Song, Lei Liu, Meihua Tan, Han Liu, Jingjing Wang, Mingzhi Ye. A high performance target-enrichment strategy for liquid biopsy [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 3407.