The Biospecimen Research Database

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%.