Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1
DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-der...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/9/4586 |
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| author | Natalia V. Mitiushkina Grigory A. Yanus Ekatherina Sh. Kuligina Tatiana A. Laidus Alexandr A. Romanko Maksim M. Kholmatov Alexandr O. Ivantsov Svetlana N. Aleksakhina Evgeny N. Imyanitov |
| author_facet | Natalia V. Mitiushkina Grigory A. Yanus Ekatherina Sh. Kuligina Tatiana A. Laidus Alexandr A. Romanko Maksim M. Kholmatov Alexandr O. Ivantsov Svetlana N. Aleksakhina Evgeny N. Imyanitov |
| author_sort | Natalia V. Mitiushkina |
| collection | DOAJ |
| description | DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5′/3′-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired <i>t</i>-test <i>p</i>-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA. |
| first_indexed | 2024-03-10T04:07:07Z |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T04:07:07Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-4a0c1b7ad30142a6836a79f6cd5a3cff2023-11-23T08:19:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01239458610.3390/ijms23094586Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1Natalia V. Mitiushkina0Grigory A. Yanus1Ekatherina Sh. Kuligina2Tatiana A. Laidus3Alexandr A. Romanko4Maksim M. Kholmatov5Alexandr O. Ivantsov6Svetlana N. Aleksakhina7Evgeny N. Imyanitov8Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDepartment of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St.-Petersburg, RussiaDNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5′/3′-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired <i>t</i>-test <i>p</i>-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.https://www.mdpi.com/1422-0067/23/9/4586duplex sequencingFFPEBotSeqSnuclease P1colorectal carcinomatumor mutation load |
| spellingShingle | Natalia V. Mitiushkina Grigory A. Yanus Ekatherina Sh. Kuligina Tatiana A. Laidus Alexandr A. Romanko Maksim M. Kholmatov Alexandr O. Ivantsov Svetlana N. Aleksakhina Evgeny N. Imyanitov Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 International Journal of Molecular Sciences duplex sequencing FFPE BotSeqS nuclease P1 colorectal carcinoma tumor mutation load |
| title | Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 |
| title_full | Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 |
| title_fullStr | Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 |
| title_full_unstemmed | Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 |
| title_short | Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 |
| title_sort | preparation of duplex sequencing libraries for archival paraffin embedded tissue samples using single strand specific nuclease p1 |
| topic | duplex sequencing FFPE BotSeqS nuclease P1 colorectal carcinoma tumor mutation load |
| url | https://www.mdpi.com/1422-0067/23/9/4586 |
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