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

Author(s): McDonough SJ, Bhagwate A, Sun Z, Wang C, Zschunke M, Gorman JA, Kopp KJ, Cunningham JM

Publication: PLoS One, 2019, Vol. 14, Page e0211400

PubMed ID: 30973937 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated differences between DNA extraction methods on the yield of DNA, percent dsDNA, and fragment size and compared extraction and library preparation methods on NGS metrics and genotype concordance. The effects of tissue type were also investigated.  Nine 10 µm sections were cut from each of 12 archival FFPE blocks including both normal and pathological breast, colon, lung, and pancreas specimens; two normal tonsil tissues; a brain stem specimen; and a cerebellum specimen. Specimens were fixed for more than 6 h before paraffin-embedding but other details of specimen procurement and FFPE processing were not specified. Sections were placed on slides and then scraped into microcentrifuge tubes. Sections were deparaffinized using the QIagen deparaffinization solution, xylene (for QIAamp extractions), or the KAPA Express Extract reagents (for KAPA express extractions). DNA was extracted using manual kits (KAPA Express Extract Kit, Promega Reliaprep FFPE gDNA Miniprep System, QIAamp FFPE tissue Kit with extention of lysis to overnight, or QIAGEN GeneRead DNA FFPE Kit) or automated kits (QIAGEN QIAsymphony DNA Mini Kit, QIAGEN GeneRead DNA FFPE Kit, Maxwell RSC DNA FFPE Kit, PerkinElmer Chemagic FFPE DNA kit, or Applied Biosystem’s Duo Mag-MAX FFPE DNA Isolation Kit). DNA was quantified using the Qubit dsDNA BR Assay and spectrophotmetery. For specimens with measurements below 2.0 ng, the concentration was verified using the Qubit dsDNA HS Assay. Fragment lengths were determined using the Analytical Fragment Analyzer High-Sensitivity Large Fragment Analysis Kit and multiplex PCR. Libraries were constructed using NEBNext Ultra II DNA Library Prep and ThruPLEX DNA-seq Kit from DNA extracted with the QIAcube GeneRead DNA FFPE Kit (manual and automated), QIAamp DNA Mini Kit, or KingFisher Duo MagMAX FFPE DNA Isolation Kit and sequenced on an Illumina HiSeq 4000. The QIAGEN QIAseq Targeted Human Comprehensive Cancer Panel and the Human Breast Cancer panels were also sequenced on these specimens using a HiSeq 4000. Statistical analyses were performed but comparisons made were not always clear.

   Summary of Findings:

   DNA yields were highest from tonsil specimens and lowest from brain stem and breast specimens, but there was no consistent effect of extraction method on yield. As expected, tumors yielded more DNA than the respective normal tissue. The percentage of DNA that was double stranded was highest using the QIAamp DNA Mini Kit, the GeneRead DNA FFPE Kit (automated or manual), the QIAsymphony DSP DNA Mini Kit, and the KingFisher Duo MagMAX FFPE DNA Isolation Kit. The ratio of the absorbance at 260 to 280 was above 1.8 for all specimens with the exception of those extracted with the KAPA Express Extract Kit and Reliaprep FFPE gDNA Miniprep System. Fragment length analysis showed the most degradation in cerebellum specimens and when the KAPA Express Extract Kit, Reliaprep FFPE gDNA Miniprep System, Maxwell RSC DNA FFPE Kit, or Chemagic MSM1 FFPE DNA Kit were used.  Highly variable results were observed when the QIAsymphony DSP DNA Mini Kit was used. The most consistently intact DNA (highest DQN) was obtained using QIAamp DNA Mini, QIAcube GeneRead DNA FFPE, and KingFisher Duo MagMAX FFPE DNA Isolation kits. Multiplex PCR confirmed the lower integrity of specimens extracted using the KAPA Express Extract, Chemagic MSM1 FFPE DNA, and QIAsymphony DSP DNA Mini kits and better amplification of longer fragments when extraction was with QIAcube GeneRead DNA FFPE Kit or the KingFisher Duo MagMAX FFPE DNA Isolation Kit.

   As expected, the most degraded specimens (cerebellum) resulted in lower mean coverage and higher duplication rates than observed for other tissue types. Using Ultra II rather than ThruPLEX for library preparation resulted in higher mean coverage (mean 25.88X versus 21.5X) and lower duplication rate (11.9% versus 26.21%). There were no clear differences between extraction method on the mean coverage or duplication rate and the concordance in genotype between extraction kits was >95% for all extraction kit combinations and both library preparation methods.

   Using the QIAseq Human Comprehensive Cancer panel, higher average molecular tag coverage was found in specimens extracted using the KingFisher Duo MagMAX FFPE DNA Isolation Kit (820.91X) than with the automated or manual QIAcube GeneRead DNA FFPE Kit (733.77X and 650.96X, respectively) or the QIAamp DNA Mini Kit (599.68X). Similarly, coverage was higher using the Breast Cancer panel for specimens extracted using the KingFisher Duo MagMAX FFPE DNA Isolation Kit (1609.23X) than with the QIAamp DNA Mini Kit (1089.31X) or the manual or automated QIAcube GeneRead DNA FFPE Kit (749.12X and 731.92X, respectively). Importantly, the average concordance in genotype calls between extraction methods was 99.59%. Finally, the lowest percentage of C>T/G>A occurred when extraction was with the QIAcube GeneRead DNA FFPE Kit (36.64%) or KingFisher Duo MagMAX FFPE DNA Isolation Kit (36.59%) and the highest when extraction was with the QIAamp DNA Mini Kit (38.06%).

   Biospecimens

   • Tissue - Brain
   • Tissue - Breast
   • Tissue - Tonsil
   • Tissue - Lung
   • Tissue - Pancreas
   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	PCR
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	KAPA Express Extract kit Promega Reliaprep FFPE gDNA Miniprep system QIAamp FFPE tissue kit (extending lysis to overnight) Manual QIAGEN GeneRead DNA FFPE kit Automated QIAGEN GeneRead DNA FFPE kit QIAGEN QIAsymphony DNA mini kit Maxwell RSC DNA FFPE Kit PerkinElmer chemagic FFPE DNA kit Applied Biosystem’s Duo Mag-MAX FFPE DNA Isolation Kit
   Biospecimen Acquisition	Biospecimen location	Brain stem Cerebellum Tonsil Breast Lung Colon Pancreas Tumor Normal
   Next generation sequencing Specific	Technology platform	QIAseq Targeted Human Comprehensive Cancer Panel Human Breast Cancer panels NEBNext Ultra II DNA Library Prep ThruPLEX DNA-seq Kit

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