Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples.

Author(s): Bonnet E, Moutet ML, Baulard C, Bacq-Daian D, Sandron F, Mesrob L, Fin B, Delépine M, Palomares MA, Jubin C, Blanché H, Meyer V, Boland A, Olaso R, Deleuze JF

Publication: PLoS One, 2018, Vol. 13, Page e0195471

PubMed ID: 29621323 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared DNA integrity, next generation sequencing performance, and genotyping between frozen and FFPE specimens and compared the use of different DNA extraction methods on results. Forty-two matched normal adjacent and tumor liver and colon specimens were obtained commercially. Of these, 26 were FFPE and 16 were frozen and all had been stored less than 3 years before DNA extraction. DNA was extracted from 10 μm sections of the FFPE specimens using Qiagen GeneRead DNA FFPE kit (10 specimens), Maxwell RSC DNA FFPE kit (8 specimens), or the QIAamp DNA FFPE Tissue kit (8 specimens). DNA was extracted from the frozen specimens using the Maxwell RSC Blood DNA kit (8 specimens) or the QIAamp DNA Micro kit (8 specimens). All extractions included an overnight Proteinase K digestion at 65˚C. DNA was quantified using Quanti-iT dsDNA assay kits and integrity determined using a TapeStation 4200. DNA exomes were captured using the Agilent Sureselect All Exons Human V5 kit and sequenced on a HiSeq2000 with 100 bp paired-end reads. As read counts varied among the specimens all data was resampled to 80 million reads. For analysis, frozen and FFPE specimens were paired according to tissue type such that those extracted with QIAamp DNA Micro kit were compared with those extracted using QIAamp DNA FFPE Tissue kit and GeneRead DNA FFPE kit and those extracted with the Maxwell RSC Blood DNA kit were compared with those extracted with Maxwell RSC DNA FFPE kit.

   Summary of Findings:

   DNA integrity numbers and median fragment length were lower for FFPE than frozen specimens (P=2.2 x 10^-16 and P=3.7 x 10^-10, respectively) and the DNA integrity and fragment length among the FFPE specimens varied significantly between extraction methods (P=1.03 x 10^-5 and P=2.2 x 10^-6, respectively). DNA extracted using the QIAamp kit had the highest integrity numbers and longest median fragment length (1622 bp) while DNA extracted using the Maxwell kit had the lowest integrity numbers and shortest median fragment length (988 bp).  

   The percentage of positions with greater than 30X coverage was higher for frozen than FFPE specimens (98.4% versus 97%, P=4.8 x 10^-7). Among FFPE specimens, the percentage of positions with greater than 30X coverage was highest in specimens extracted using QIAamp or GeneRead and lowest when extracted using the Maxwell RSC DNA FFPE kit (P=2.3 x 10^-14). The mean median coverage was higher in frozen than FFPE specimens (87.6X versus 74.3X, P=1.3 x 10^-5) and differed among FFPE extraction kits (P=6.5 x 10^-14). The mean median coverage was highest for specimens extracted using the QIAamp kit and lowest when extracted using the Maxwell kit. The percentage duplicate reads was higher for FFPE than frozen specimens (12.2% versus 7.9%, P=0.004) and differed among the extraction methods for FFPE specimens (P=6.4 x 10^-6) with the GeneRead kit producing the most duplicate reads and the Maxwell kit the fewest. The percentage of reads mapping outside the target region is lower for FFPE than frozen specimens (19.6% versus 21.3%, P=0.0002) and was lowest for FFPE specimens when QIAamp or GeneRead kits were used rather than the Maxwell kit (19.1% and 19.6%, versus 21.4%).

   More SNVs and INDELs were found in frozen specimens than FFPE specimens (40,938 and 3680 versus 39,748 and 3370, P=0.0003 and P=1.7 x10^-5). When filtered to only consider SNVs and INDELs identified in both the FFPE and frozen specimens, 99.98-99.99% of SNVs and 98.44% of INDELs were concordant. The difference in number of variants between frozen and FFPE specimens was significantly different among the extraction kits (P=3.9 x 10^-12) and was lowest when the GeneRead kit was used. As expected, the C>T and G>A mutations occurred more often in FFPE than frozen specimens (P=0.0005) and occurred most often when extraction was with the Maxwell kit (P=0.009). Of the tumor-specific SNVs in colon specimens, 36% of those identified in FFPE specimens overlapped with those in frozen specimens. Similarly, 16% of the tumor specific SNVs in FFPE liver specimens were also found in frozen liver specimens. Importantly, analysis of top genes in liver and colon cancer showed greater than 30X coverage for all genes in frozen and FFPE specimens, except in the case of PIK3CA in FFPE specimens.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Liver

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA FFPE Tissue kit GeneRead DNA FFPE kit Maxwell RSC DNA FFPE Kit
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen

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