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Isolation and next generation sequencing of archival formalin-fixed DNA

Lookup NU author(s): Dr Ahlam AlqahtaniORCiD, Andrew Skelton, Dr Lorraine EleyORCiD, Dr Srinivas Annavarapu, Professor Deborah HendersonORCiD, Dr Bill ChaudhryORCiD

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2020 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society. 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.

Publication metadata

Author(s): Alqahtani A, Skelton A, Eley L, Annavarapu S, Henderson DJ, Chaudhry B

Publication type: Article

Publication status: Published

Journal: Journal of Anatomy

Year: 2020

Volume: 237

Issue: 3

Pages: 587-600

Print publication date: 01/09/2020

Online publication date: 19/05/2020

Acceptance date: 07/04/2020

Date deposited: 02/06/2020

ISSN (print): 0021-8782

ISSN (electronic): 1469-7580

Publisher: John Wiley & Sons Ltd

URL: https://doi.org/10.1111/joa.13209

DOI: 10.1111/joa.13209

PubMed id: 32426881

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