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      Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods

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          Abstract

          Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS.

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          Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine

          Translating whole exome sequencing (WES) for prospective clinical use may impact the care of cancer patients; however, multiple innovations are necessary for clinical implementation. These include: (1) rapid and robust WES from formalin-fixed paraffin embedded (FFPE) tumor tissue, (2) analytical output similar to data from frozen samples, and (3) clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival FFPE tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a “long tail” of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15/16 patients. In one patient, previously undetected findings guided clinical trial enrollment leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.
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            Ancient DNA.

            DNA that has been recovered from archaeological and palaeontological remains makes it possible to go back in time and study the genetic relationships of extinct organisms to their contemporary relatives. This provides a new perspective on the evolution of organisms and DNA sequences. However, the field is fraught with technical pitfalls and needs stringent criteria to ensure the reliability of results, particularly when human remains are studied.
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              DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.

              We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, 'jumping PCR', i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than approximately 0.1% even under the unlikely scenario where each amplification starts from a single template molecule.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysis
                Role: Formal analysis
                Role: Methodology
                Role: Supervision
                Role: Supervision
                Role: ConceptualizationRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 April 2019
                2019
                : 14
                : 4
                : e0211400
                Affiliations
                [1 ] Medical Genome Facility, Mayo Clinic, Rochester, Minnesota, United States of America
                [2 ] Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota, United States of America
                [3 ] Pathology Research Core, Mayo Clinic, Rochester, Minnesota, United States of America
                [4 ] Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, Minnesota, United States of America
                University of Helsinki, FINLAND
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-2638-3081
                http://orcid.org/0000-0002-8159-3025
                Article
                PONE-D-19-00906
                10.1371/journal.pone.0211400
                6459541
                30973937
                aa220b33-bd8f-4043-8ab7-4d14018a1071
                © 2019 McDonough et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 January 2019
                : 20 March 2019
                Page count
                Figures: 5, Tables: 4, Pages: 15
                Funding
                This work was supported by the Mayo Clinic Center for Individualized Medicine. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and analysis methods
                Extraction techniques
                DNA extraction
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Biology and Life Sciences
                Anatomy
                Brain
                Cerebral Cortex
                Cerebellum
                Medicine and Health Sciences
                Anatomy
                Brain
                Cerebral Cortex
                Cerebellum
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Biomolecular isolation
                DNA isolation
                Research and analysis methods
                Molecular biology techniques
                Biomolecular isolation
                DNA isolation
                Biology and Life Sciences
                Genetics
                Heredity
                Genetic Mapping
                Variant Genotypes
                Biology and life sciences
                Genetics
                DNA
                DNA libraries
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA libraries
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Biology and Life Sciences
                Anatomy
                Endocrine System
                Pancreas
                Medicine and Health Sciences
                Anatomy
                Endocrine System
                Pancreas
                Biology and Life Sciences
                Anatomy
                Exocrine Glands
                Pancreas
                Medicine and Health Sciences
                Anatomy
                Exocrine Glands
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                Custom metadata
                Data is available in the public repository Sequence Read Archive https://www.ncbi.nlm.nih.gov/sra PRJNA5260131.

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