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Lookup NU author(s): Dr Ahlam Alqahtani, Andrew Skelton, Dr Lorraine Eley, Dr Srinivas Annavarapu, Professor Deborah HendersonORCiD, Dr Bill Chaudhry
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 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.
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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