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

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...

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Autores principales: De Luca, Giuseppa, Cardinali, Barbara, Del Mastro, Lucia, Lastraioli, Sonia, Carli, Franca, Ferrarini, Manlio, Calin, George A., Garuti, Anna, Mazzitelli, Carlotta, Zupo, Simona, Dono, Mariella
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352435/
https://www.ncbi.nlm.nih.gov/pubmed/32575430
http://dx.doi.org/10.3390/ijms21124364
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author De Luca, Giuseppa
Cardinali, Barbara
Del Mastro, Lucia
Lastraioli, Sonia
Carli, Franca
Ferrarini, Manlio
Calin, George A.
Garuti, Anna
Mazzitelli, Carlotta
Zupo, Simona
Dono, Mariella
author_facet De Luca, Giuseppa
Cardinali, Barbara
Del Mastro, Lucia
Lastraioli, Sonia
Carli, Franca
Ferrarini, Manlio
Calin, George A.
Garuti, Anna
Mazzitelli, Carlotta
Zupo, Simona
Dono, Mariella
author_sort De Luca, Giuseppa
collection PubMed
description 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.
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spelling pubmed-73524352020-07-15 Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling De Luca, Giuseppa Cardinali, Barbara Del Mastro, Lucia Lastraioli, Sonia Carli, Franca Ferrarini, Manlio Calin, George A. Garuti, Anna Mazzitelli, Carlotta Zupo, Simona Dono, Mariella Int J Mol Sci Article 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. MDPI 2020-06-19 /pmc/articles/PMC7352435/ /pubmed/32575430 http://dx.doi.org/10.3390/ijms21124364 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
De Luca, Giuseppa
Cardinali, Barbara
Del Mastro, Lucia
Lastraioli, Sonia
Carli, Franca
Ferrarini, Manlio
Calin, George A.
Garuti, Anna
Mazzitelli, Carlotta
Zupo, Simona
Dono, Mariella
Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling
title Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling
title_full Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling
title_fullStr Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling
title_full_unstemmed Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling
title_short Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling
title_sort optimization of a wga-free molecular tagging-based ngs protocol for ctcs mutational profiling
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352435/
https://www.ncbi.nlm.nih.gov/pubmed/32575430
http://dx.doi.org/10.3390/ijms21124364
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