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Isolation and next generation sequencing of archival formalin‐fixed DNA
DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin‐fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil‐N‐glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of geno...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476199/ https://www.ncbi.nlm.nih.gov/pubmed/32426881 http://dx.doi.org/10.1111/joa.13209 |
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author | Alqahtani, Ahlam Skelton, Andrew Eley, Lorraine Annavarapu, Srinivas Henderson, Deborah J. Chaudhry, Bill |
author_facet | Alqahtani, Ahlam Skelton, Andrew Eley, Lorraine Annavarapu, Srinivas Henderson, Deborah J. Chaudhry, Bill |
author_sort | Alqahtani, Ahlam |
collection | PubMed |
description | DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin‐fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil‐N‐glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin‐fixed human tissues. When compared with a leading commercial formalin‐fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub‐cloning and Sanger sequencing from UNG‐treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte‐derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin‐fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four‐fold to 56% and doubled specificity to 68% when compared with a standard hard‐filtering approach. Thus, high‐quality DNA can be extracted from excessively formalin‐fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub‐cloned amplicons is an important methodological step in assessing DNA quality. |
format | Online Article Text |
id | pubmed-7476199 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-74761992020-09-11 Isolation and next generation sequencing of archival formalin‐fixed DNA Alqahtani, Ahlam Skelton, Andrew Eley, Lorraine Annavarapu, Srinivas Henderson, Deborah J. Chaudhry, Bill J Anat Methods DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin‐fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil‐N‐glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin‐fixed human tissues. When compared with a leading commercial formalin‐fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub‐cloning and Sanger sequencing from UNG‐treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte‐derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin‐fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four‐fold to 56% and doubled specificity to 68% when compared with a standard hard‐filtering approach. Thus, high‐quality DNA can be extracted from excessively formalin‐fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub‐cloned amplicons is an important methodological step in assessing DNA quality. John Wiley and Sons Inc. 2020-05-19 2020-09 /pmc/articles/PMC7476199/ /pubmed/32426881 http://dx.doi.org/10.1111/joa.13209 Text en © 2020 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Socity This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Alqahtani, Ahlam Skelton, Andrew Eley, Lorraine Annavarapu, Srinivas Henderson, Deborah J. Chaudhry, Bill Isolation and next generation sequencing of archival formalin‐fixed DNA |
title | Isolation and next generation sequencing of archival formalin‐fixed DNA |
title_full | Isolation and next generation sequencing of archival formalin‐fixed DNA |
title_fullStr | Isolation and next generation sequencing of archival formalin‐fixed DNA |
title_full_unstemmed | Isolation and next generation sequencing of archival formalin‐fixed DNA |
title_short | Isolation and next generation sequencing of archival formalin‐fixed DNA |
title_sort | isolation and next generation sequencing of archival formalin‐fixed dna |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476199/ https://www.ncbi.nlm.nih.gov/pubmed/32426881 http://dx.doi.org/10.1111/joa.13209 |
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