Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Standardization of the preanalytical phase of DNA extraction from fixed tissue for next-generation sequencing analyses.

Author(s): Kofanova O, Bellora C, Garcia Frasquilho S, Antunes L, Hamot G, Mathay C, Mommaerts K, Muller A, DeWitt B, Betsou F

Publication: N Biotechnol, 2019, Vol. , Page

PubMed ID: 31398512 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare DNA yield, purity, and integrity and NGS data from FFPE, PFPE, and RNAlater preserved specimens and to compare results using different DNA extraction methods. The DNA yield and integrity assays were used to develop a DNA quality score that predicted NGS success. Matched FFPE, PFPE, and RNAlater-preserved specimens from three breast tumors, three normal colons, three normal skin specimens, two kidney tumors, one normal kidney, one uterine tumor, and two normal uterus specimens (15 specimens total). All tumor specimens contained 40-90% tumor, 5-10% normal tissue, and <20% necrosis. The authors report specimen preservation was according to manufacturers’ protocols. DNA was extracted from RNAlater preserved specimens using the DNeasy Blood &Tissue kit. DNA was extracted from two to three 20µm scrolls of FFPE and PFPE specimens by: (1) deparaffinization in xylene followed by two absolute ethanol washes and extraction using the DNeasy Blood & Tissue Kit or (2) deparaffinization in Chemagic lysis buffer at 95°C followed by extraction using the Chemagic DNA Tissue Kit special: from FFPE specimens by (3) deparaffinization using three xylene washes, two methanol washes and a graded ethanol series followed by the QIAamp DNA FFPE Tissue Kit, and from PFPE specimens using (4) a single xylene wash followed by a single absolute ethanol wash and extraction with PAXgene Tissue DNA kit. DNA concentration was determined spectrophotometrically and using Quant-IT PicoGreen dsDNA Assay Kit, purity was evaluated spectrophotometrically, and integrity was assessed by GAPDH multiplex PCR (100, 200, 300, and 400 bp). DNA integrity was also assessed based on the yield using the BIOSCORE Screening and Amplification Kit with DNA scored as poor (<1 µg generated) intermediate (1-3 µg generated), good (3-10 µg generated), or excellent (>10 µg generated). Targeted TruSeq Amplicon Cancer Panel libraries were constructed following the Illumina protocol using 150 ng DNA (FFPE specimens) or 250 ng DNA (RNAlater and PFPE specimens) from eight patients and sequenced using a MiSEq instrument. Analysis was conducted using the Illumina Variant Studio software. Acceptance for the run was defined as having a global Q score of 30-80%, a raw cluster density between 500-1000 k/mm², and >85% of clusters passing filters. Samples were considered acceptable if Phred score was >49, read depth was >999, and alternate variant frequency was >5%.

   Summary of Findings:

   As expected, the percentage of dsDNA was lower in FFPE and PFPE specimens than RNAlater specimens (mean of <30% versus mean of 60%), regardless of extraction method. The percentage of dsDNA from FFPE specimens was significantly higher when extraction was with the QIAamp FFPE DNA kit than with the Chemagic DNA or DNeasy kits and the percentage of dsDNA from PFPE was significantly higher when extraction was with PAXgene or the Chemagic DNA kits than the DNeasy kit. DNA purity (OD260/230) did not differ based on preservative used but was lower in specimens extracted using the Chemagic Kit than the other methods. For the 15 specimens analyzed, all four amplicons (100-400 bp) were amplifiable in eight of the RNAlater specimens extracted with DNeasy, seven PFPE specimens extracted with Chemagic, one PFPE specimen extracted with PAXgene, and four FFPE specimens extracted with Chemagic. Three of the four amplicons were amplifiable in two RNAlater specimens extracted with DNeasy, three PFPE specimens extracted with Chemagic, one PFPE specimen extracted with DNeasy, and five FFPE specimens extracted with Chemagic. Two amplicons were amplifiable in one RNAlater specimen extracted with DNeasy, two PFPE specimens extracted with Chemagic, five PFPE specimens extracted with DNeasy, nine PFPE specimens extracted with PAXgene, three FFPE specimens extracted with Chemagic, one FFPE specimen extracted QIAamp or DNeasy. A single band was amplifiable from four PFPE specimens extracted with DNeasy, two PFPE specimens extracted with PAXgene, one FFPE specimen extracted with Chemagic, 11 FFPE specimens extracted with QIAamp, and one FFPE specimen extracted with DNeasy. No PCR bands were observed in two RNAlater specimens extracted with DNeasy, three PFPE specimens extracted with Chemagic, five PFPE specimens extracted with DNeasy, three PFPE specimens extracted with PAXgene, two FFPE specimens extracted with Chemagic, three FFPE specimens extracted with QIAamp, and 13 FFPE specimens extracted with DNeasy. Similarly, the integrity of the DNA as determined by the WGA assay was excellent in all 15 RNAlater specimens, seven PFPE specimens (6 Chemagic and 1 PAXgene), and only two FFPE specimens (both QIAamp); good in 20 PFPE specimens (4 Chemagic, 3 DNeasy, and 13 PAXgene) and 16 FFPE specimens (5 Chemagic and 11 QIAamp); intermediate in 18 PFPE specimens (5 Chemagic, 12 DNeasy, and 1 PAXgene) and 14 FFPE specimens (9 Chemagic, 3 DNeasy, and 2 QIAamp); and poor in 13 FFPE specimens (12 DNeasy and 1 Chemagic).

   All the specimens had >1000x coverage but the mean percentage of reads mapped was significantly lower for FFPE specimens extracted with QIAamp (91.64%), DNeasy (70.19%), and Chemagic (91.45%) than for RNAlater specimens (94.90%, P<0.05 all). A comparable percentage of the total variants were identified as Passing Filter (PF) in RNAlater and PFPE specimens, regardless of extraction method. In contrast, FFPE specimens extracted with Chemagic or DNeasy had a significantly lower percentage of the total variants identified as PF than RNAlater specimens (P<0.05). The PF variants in matched FFPE and RNAlater specimens showed varying levels of overlap that was dependent on the patient and extraction method. ROC analysis showed the best predictor of high quality NGS (>15% PF) was at least 25% dsDNA with amplification of the 200 bp fragment by multiplex PCR (2 bands) and scoring intermediate or better in the WGA assay. When the sum of the percentage dsDNA was divided by 10, number of bands amplified in multiplex PCR and WGA score (1-poor, 2-intermediate, 3-good, 4 excellent) was >7.5, the sensitivity and specificity for high quality NGS were 79% and 94%, respectively.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Breast
   • Tissue - Uterus
   • Tissue - Kidney
   • Tissue - Skin

   Preservative Types

   • Formalin
   • RNAlater
   • PAXgene

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	Whole genome amplification
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	RNAlater PAXgene Formalin (buffered)
   PCR Specific	Technology platform	WGA
   Analyte Extraction and Purification	Analyte isolation method	DNeasy Blood & Tissue Kit Chemagic DNA Tissue Kit special QIAamp DNA FFPE Tissue kit PAXgene Tissue DNA kit

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