Universität zu Köln

Kong, Say Li, Li, Huipeng, Tai, Joyce A., Courtois, Elise T., Poh, Huay Mei, Lau, Dawn Pingxi, Haw, Yu Xuan, Iyer, Narayanan Gopalakrishna, Tan, Daniel Shao Weng, Prabhakar, Shyam, Ruff, Dave and Hillmer, Axel M. (2019). Concurrent Single-Cell RNA and Targeted DNA Sequencing on an Automated Platform for Comeasurement of Genomic and Transcriptomic Signatures. Clin. Chem., 65 (2). S. 272 - 282. WASHINGTON: AMER ASSOC CLINICAL CHEMISTRY. ISSN 1530-8561

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Abstract

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

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Kong, Say Li UNSPECIFIED UNSPECIFIED UNSPECIFIED Li, Huipeng UNSPECIFIED UNSPECIFIED UNSPECIFIED Tai, Joyce A. UNSPECIFIED UNSPECIFIED UNSPECIFIED Courtois, Elise T. UNSPECIFIED UNSPECIFIED UNSPECIFIED Poh, Huay Mei UNSPECIFIED UNSPECIFIED UNSPECIFIED Lau, Dawn Pingxi UNSPECIFIED UNSPECIFIED UNSPECIFIED Haw, Yu Xuan UNSPECIFIED UNSPECIFIED UNSPECIFIED Iyer, Narayanan Gopalakrishna UNSPECIFIED UNSPECIFIED UNSPECIFIED Tan, Daniel Shao Weng UNSPECIFIED UNSPECIFIED UNSPECIFIED Prabhakar, Shyam UNSPECIFIED UNSPECIFIED UNSPECIFIED Ruff, Dave UNSPECIFIED UNSPECIFIED UNSPECIFIED Hillmer, Axel M. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-158149
DOI:	10.1373/clinchem.2018.295717
Journal or Publication Title:	Clin. Chem.
Volume:	65
Number:	2
Page Range:	S. 272 - 282
Date:	2019
Publisher:	AMER ASSOC CLINICAL CHEMISTRY
Place of Publication:	WASHINGTON
ISSN:	1530-8561
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language LUNG-CANCER; MESENCHYMAL TRANSITION; EGFR MUTATION; RESISTANCE; HETEROGENEITY; GEFITINIB; AMPLIFICATION; THERAPY; SEQ Multiple languages Medical Laboratory Technology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/15814