Abstract
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.
Highlights
Formalin provides convenient fixation and storage of human tissue samples prior to routine pathological investigation (Fox et al, 1985).It maintains tissue integrity, allows application of a range of histological stains and permits immunohistochemistry, often enhanced through antigen retrieval (Shi et al, 1991)
Unravelling the genetic basis of inherited disease is reliant on DNA, and historical formalin-fixed (FF) or formalin-fixed paraffin wax-embedded (FFPE) samples may be the only source of DNA from affected but deceased family members
We have optimised the extraction of DNA obtained from overly formalin-fixed human tissue and evaluated its performance as a template for chain termination (Sanger) sequencing and next generation sequencing (NGS)
Summary
Formalin provides convenient fixation and storage of human tissue samples prior to routine pathological investigation (Fox et al, 1985). It maintains tissue integrity, allows application of a range of histological stains and permits immunohistochemistry, often enhanced through antigen retrieval (Shi et al, 1991). Following our recent morphological study of congenitally malformed hearts (Crucean et al, 2017) we were asked by clinicians and anatomists whether this extensive collection of fully morphologically characterised organs, routinely stored in formalin, could be used to discover causative mutations by exome sequencing. We performed generation exome sequencing on myocardial tissue that had been stored for 2 years in formalin and compared the results with those obtained from NGS of DNA freshly extracted from peripheral blood from the same patient. The high rates of false-positive results in FF-DNA preclude its use as a primary source of DNA for discovery of unknown causative genes, we suggest that it may be useful as a confirmatory DNA source
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
