Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Mitiushkina, Natalia V., Yanus, Grigory A., Kuligina, Ekatherina Sh., Laidus, Tatiana A., Romanko, Alexandr A., Kholmatov, Maksim M., Ivantsov, Alexandr O., Aleksakhina, Svetlana N., Imyanitov, Evgeny N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9105346/
https://www.ncbi.nlm.nih.gov/pubmed/35562977
http://dx.doi.org/10.3390/ijms23094586
_version_ 1784708018138513408
author Mitiushkina, Natalia V.
Yanus, Grigory A.
Kuligina, Ekatherina Sh.
Laidus, Tatiana A.
Romanko, Alexandr A.
Kholmatov, Maksim M.
Ivantsov, Alexandr O.
Aleksakhina, Svetlana N.
Imyanitov, Evgeny N.
author_facet Mitiushkina, Natalia V.
Yanus, Grigory A.
Kuligina, Ekatherina Sh.
Laidus, Tatiana A.
Romanko, Alexandr A.
Kholmatov, Maksim M.
Ivantsov, Alexandr O.
Aleksakhina, Svetlana N.
Imyanitov, Evgeny N.
author_sort Mitiushkina, Natalia V.
collection PubMed
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 t-test p-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.
format Online
Article
Text
id pubmed-9105346
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-91053462022-05-14 Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1 Mitiushkina, Natalia V. Yanus, Grigory A. Kuligina, Ekatherina Sh. Laidus, Tatiana A. Romanko, Alexandr A. Kholmatov, Maksim M. Ivantsov, Alexandr O. Aleksakhina, Svetlana N. Imyanitov, Evgeny N. Int J Mol Sci Article 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 t-test p-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. MDPI 2022-04-21 /pmc/articles/PMC9105346/ /pubmed/35562977 http://dx.doi.org/10.3390/ijms23094586 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Mitiushkina, Natalia V.
Yanus, Grigory A.
Kuligina, Ekatherina Sh.
Laidus, Tatiana A.
Romanko, Alexandr A.
Kholmatov, Maksim M.
Ivantsov, Alexandr O.
Aleksakhina, Svetlana N.
Imyanitov, Evgeny N.
Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9105346/
https://www.ncbi.nlm.nih.gov/pubmed/35562977
http://dx.doi.org/10.3390/ijms23094586
work_keys_str_mv AT mitiushkinanataliav preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT yanusgrigorya preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT kuliginaekatherinash preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT laidustatianaa preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT romankoalexandra preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT kholmatovmaksimm preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT ivantsovalexandro preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT aleksakhinasvetlanan preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1
AT imyanitovevgenyn preparationofduplexsequencinglibrariesforarchivalparaffinembeddedtissuesamplesusingsinglestrandspecificnucleasep1