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Open AccessArticle

Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling

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Molecular Diagnostic Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy
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Breast Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy
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Department of Internal Medicine (Di.M.I.), University of Genova, 16132 Genova, Italy
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Department of Pathology, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy
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DIMES, Anatomy Section, Medical School, University of Genova, 16132 Genova, Italy
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Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, TX 77030, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(12), 4364; https://doi.org/10.3390/ijms21124364
Received: 24 April 2020 / Revised: 11 June 2020 / Accepted: 17 June 2020 / Published: 19 June 2020
(This article belongs to the Section Molecular Oncology)
Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use. View Full-Text
Keywords: Circulating Tumor Cells; breast cancer; liquid biopsy; Next-Generation Sequencing; molecular tagging; Whole Genome Amplification-free; single-cell genomics; biomarkers; precision medicine Circulating Tumor Cells; breast cancer; liquid biopsy; Next-Generation Sequencing; molecular tagging; Whole Genome Amplification-free; single-cell genomics; biomarkers; precision medicine
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De Luca, G.; Cardinali, B.; Del Mastro, L.; Lastraioli, S.; Carli, F.; Ferrarini, M.; Calin, G.A.; Garuti, A.; Mazzitelli, C.; Zupo, S.; Dono, M. Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling. Int. J. Mol. Sci. 2020, 21, 4364.

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