Journal
CLINICAL CHEMISTRY
Volume 65, Issue 2, Pages 272-281Publisher
AMER ASSOC CLINICAL CHEMISTRY
DOI: 10.1373/clinchem.2018.295717
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Funding
- Biomedical Research Council, Singapore [SPF2012/003, H16/99/b0/003]
- National Medical Research Council, Singapore [NMRC/OFYIRG/0056/2017]
- Career Development Award by the Joint Council Office (JCO), Agency for Science, Technology and Research (A*STAR), Singapore [14302FG096]
- Genome Institute of Singapore
- Translational & Clinical Research Program by the National Medical Research Council (NMRC) [NMRC/TCR/007-NCC/2013]
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BACKGROUND: The comeasurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information correlates with the transcriptomic phenotype. However, existing technologies have low throughput and laborious work flows. METHODS: We developed a new method for concurrent sequencing of the transcriptome and 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 transcriptome and 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 variations and haplotype construction. We detected expression signatures of tyrosine kinase inhibitor (TKI) resistance, epidermal growth factor receptor (EGFR) amplification, and expansion of the T790M mutation 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 transcriptomic changes in single cells. It is operated on an automated, commercially available single-cell isolation platform, making its implementation straightforward. (C) 2018 American Association for Clinical Chemistry
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