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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...

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Autores principales: Alqahtani, Ahlam, Skelton, Andrew, Eley, Lorraine, Annavarapu, Srinivas, Henderson, Deborah J., Chaudhry, Bill
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
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.
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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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