Isolation and next generation sequencing of archival formalin-fixed DNA.

Ahlam Alqahtani, Andrew Skelton, Lorraine Eley, Srinivas Annavarapu, Deborah J Henderson, Bill Chaudhry

Journal of anatomy 2020 Sep

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

Citation

Ahlam Alqahtani, Andrew Skelton, Lorraine Eley, Srinivas Annavarapu, Deborah J Henderson, Bill Chaudhry. Isolation and next generation sequencing of archival formalin-fixed DNA. Journal of anatomy. 2020 Sep;237(3):587-600