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Volume 22
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Communication

Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development

by
Elizabeth A. Mickler
1,
Huaxin Zhou
1,
Tzu L. Phang
2,
Mark W. Geraci
3,
Robert S. Stearman
1 and
Catherine R. Sears
1,4,*
1
Division of Pulmonary, Critical Care, Sleep and Occupational Medicine, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
2
Division of Biomedical Informatics and Personalized Medicine, Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045, USA
3
Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
4
The Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN 46202, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(21), 12034; https://doi.org/10.3390/ijms222112034
Submission received: 24 September 2021 / Revised: 22 October 2021 / Accepted: 31 October 2021 / Published: 6 November 2021
(This article belongs to the Collection Feature Papers in Molecular Biophysics)
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Abstract

Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer.
Keywords: lung cancer; carcinogenesis; mouse; Kras; copy number alterations lung cancer; carcinogenesis; mouse; Kras; copy number alterations

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MDPI and ACS Style

Mickler, E.A.; Zhou, H.; Phang, T.L.; Geraci, M.W.; Stearman, R.S.; Sears, C.R. Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development. Int. J. Mol. Sci. 2021, 22, 12034. https://doi.org/10.3390/ijms222112034

AMA Style

Mickler EA, Zhou H, Phang TL, Geraci MW, Stearman RS, Sears CR. Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development. International Journal of Molecular Sciences. 2021; 22(21):12034. https://doi.org/10.3390/ijms222112034

Chicago/Turabian Style

Mickler, Elizabeth A., Huaxin Zhou, Tzu L. Phang, Mark W. Geraci, Robert S. Stearman, and Catherine R. Sears. 2021. "Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development" International Journal of Molecular Sciences 22, no. 21: 12034. https://doi.org/10.3390/ijms222112034

APA Style

Mickler, E. A., Zhou, H., Phang, T. L., Geraci, M. W., Stearman, R. S., & Sears, C. R. (2021). Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development. International Journal of Molecular Sciences, 22(21), 12034. https://doi.org/10.3390/ijms222112034

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