Use of FFPE-derived DNA in next generation sequencing

DNA extraction methods

Samantha J. Mcdonough, Aditya Bhagwate, Zhifu Sun, Chen Wang, Michael Zschunke, Joshua A. Gorman, Karla J. Kopp, Julie M. Cunninghamid

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere0211400
JournalPloS one
Volume14
Issue number4
DOIs
StatePublished - Apr 1 2019

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DNA Sequence Analysis
formalin
Paraffin
alkanes
Formaldehyde
sequence analysis
DNA
Tissue
Alcedinidae
Exome
methodology
Multiplex Polymerase Chain Reaction
Gene Library
Libraries
Medical Records
Acquired Immunodeficiency Syndrome
Genotype
genomics
Polymerase Chain Reaction
tissues

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Mcdonough, S. J., Bhagwate, A., Sun, Z., Wang, C., Zschunke, M., Gorman, J. A., ... Cunninghamid, J. M. (2019). Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods. PloS one, 14(4), [e0211400]. https://doi.org/10.1371/journal.pone.0211400

Use of FFPE-derived DNA in next generation sequencing : DNA extraction methods. / Mcdonough, Samantha J.; Bhagwate, Aditya; Sun, Zhifu; Wang, Chen; Zschunke, Michael; Gorman, Joshua A.; Kopp, Karla J.; Cunninghamid, Julie M.

In: PloS one, Vol. 14, No. 4, e0211400, 01.04.2019.

Research output: Contribution to journalArticle

Mcdonough, SJ, Bhagwate, A, Sun, Z, Wang, C, Zschunke, M, Gorman, JA, Kopp, KJ & Cunninghamid, JM 2019, 'Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods', PloS one, vol. 14, no. 4, e0211400. https://doi.org/10.1371/journal.pone.0211400
Mcdonough, Samantha J. ; Bhagwate, Aditya ; Sun, Zhifu ; Wang, Chen ; Zschunke, Michael ; Gorman, Joshua A. ; Kopp, Karla J. ; Cunninghamid, Julie M. / Use of FFPE-derived DNA in next generation sequencing : DNA extraction methods. In: PloS one. 2019 ; Vol. 14, No. 4.
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