Concurrent Single-Cell RNA and Targeted DNA Sequencing on an Automated Platform for Comeasurement of Genomic and Transcriptomic Signatures

Background: The comeasurement of both genomic and transcriptomicsignatures in single cells is of fundamental importance to accurately assess how the genetic information correlates with the transcriptomicphenotype. However, existing technologies have low throughput and laborious work flows. Methods: We developed a new method for concurrent sequencing of the transcriptomeand targeted genomic regions (CORTAD-seq) within the same single cell on an automated microfluidic platform. The method was compatible with the downstream library preparation, allowing easy integration into existing next-generation sequencing work flows. We incorporated a single-cell bioinformatics pipeline for transcriptomeand mutation analysis. Results: As proof of principle, we applied CORTAD-seq to lung cancer cell lines to dissect the cellular consequences of mutations that result in resistance to targeted therapy. We obtained a mean detection of 6000 expressed genes and an exonic rate of 50%. The targeted DNA-sequencing data achieved a 97.8% detection rate for mutations and allowed for the identification of copy number variationsand haplotype construction. We detected expression signatures of tyrosine kinase inhibitor (TKI) resistance, epidermal growth factor receptor ( EGFR ) amplification, and expansion of the T790Mmutation among resistant cells. We also identified characteristics for TKI resistance that were independent of EGFR T790M, indicating that other alterations are required for resistance in this context. Conclusions: CORTAD-seq allows assessment of the interconnection between genetic and transcriptomicchanges in single cells. It is operated on an automated, commercially available single-cell isolation platform, making its implementation straightforward.