Abstract 439: Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells
Authors: Ericson Nolan G. , Ramirez Arturo B. , Clein Alisa C. , Higano Celestia S. , Sabath Daniel E. , Kaldjian Eric P. 
Published: August 2019

Abstract
Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel.
Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite.
Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was &lt; 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis.
Conclusions. Amplicon-based targeted single-cell sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs.
Citation Format: Nolan G. Ericson, Arturo B. Ramirez, Alisa C. Clein, Celestia S. Higano, Daniel E. Sabath, Eric P. Kaldjian. Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 439.