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Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568385/ https://www.ncbi.nlm.nih.gov/pubmed/31199826 http://dx.doi.org/10.1371/journal.pone.0217724 |
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author | Akahane, Toshiaki Yamaguchi, Tomomi Kato, Yasutaka Yokoyama, Seiya Hamada, Taiji Nishida, Yukari Higashi, Michiyo Nishihara, Hiroshi Suzuki, Shinsuke Ueno, Shinichi Tanimoto, Akihide |
author_facet | Akahane, Toshiaki Yamaguchi, Tomomi Kato, Yasutaka Yokoyama, Seiya Hamada, Taiji Nishida, Yukari Higashi, Michiyo Nishihara, Hiroshi Suzuki, Shinsuke Ueno, Shinichi Tanimoto, Akihide |
author_sort | Akahane, Toshiaki |
collection | PubMed |
description | In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 10(3) or 2 × 10(4) cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 10(3) cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine. |
format | Online Article Text |
id | pubmed-6568385 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-65683852019-06-20 Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis Akahane, Toshiaki Yamaguchi, Tomomi Kato, Yasutaka Yokoyama, Seiya Hamada, Taiji Nishida, Yukari Higashi, Michiyo Nishihara, Hiroshi Suzuki, Shinsuke Ueno, Shinichi Tanimoto, Akihide PLoS One Research Article In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 10(3) or 2 × 10(4) cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 10(3) cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine. Public Library of Science 2019-06-14 /pmc/articles/PMC6568385/ /pubmed/31199826 http://dx.doi.org/10.1371/journal.pone.0217724 Text en © 2019 Akahane et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Akahane, Toshiaki Yamaguchi, Tomomi Kato, Yasutaka Yokoyama, Seiya Hamada, Taiji Nishida, Yukari Higashi, Michiyo Nishihara, Hiroshi Suzuki, Shinsuke Ueno, Shinichi Tanimoto, Akihide Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
title | Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
title_full | Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
title_fullStr | Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
title_full_unstemmed | Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
title_short | Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
title_sort | comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568385/ https://www.ncbi.nlm.nih.gov/pubmed/31199826 http://dx.doi.org/10.1371/journal.pone.0217724 |
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