학술논문
Effects of Improved DNA Integrity by Punch From Tissue Blocks as Compared to Pinpoint Extraction From Unstained Slides on Next-Generation Sequencing Quality Metrics
Original Article
Original Article
Document Type
Report
Author
Source
American Journal of Clinical Pathology. July 2019, Vol. 152 Issue 1, p27, 9 p.
Subject
Language
English
ISSN
0002-9173
Abstract
Next-generation sequencing (NGS) is changing the landscape of clinical molecular genetic diagnostics. (1) The reliability of NGS performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples depends on adherence to strict preanalytical [...]
Objectives: To compare the effects of two methods of formalin-fixed paraffin-embedded (FFPE) tissue harvesting on DNA quality and next-generation sequencing (NGS) quality metrics. Methods: DNA integrity number (DIN) and NGS quality metrics resulting from DNA extraction and sequencing of 199 sequential samples harvested via the Pinpoint Slide DNA Isolation System and the punch method were compared. Results: DNA extracted from FFPE tissue punches had higher DIN than that extracted from Pinpoint samples (mean [+ or -] SD, 6.18 [+ or -] 0.83 vs 5.09 [+ or -] 0.91; P < .0001), indicating less degradation. Lower DIN correlated with lower-quality metrics of NGS, that is lower percentage of unique on-target reads, average depth of coverage, and percentage of positions with coverage depth greater than or equal to 100x, 400x, and 1,000x. Conclusions: Our study demonstrated methods to harvest tissue from FFPE blocks may affect quality of DNA, which in turn has an effect on other NGS quality metrics. Key Words: Next-generation sequencing; Tissue harvesting; Formalin-fixed paraffin-embedded; DNA integrity number; Quality metrics; Preanalytical
Objectives: To compare the effects of two methods of formalin-fixed paraffin-embedded (FFPE) tissue harvesting on DNA quality and next-generation sequencing (NGS) quality metrics. Methods: DNA integrity number (DIN) and NGS quality metrics resulting from DNA extraction and sequencing of 199 sequential samples harvested via the Pinpoint Slide DNA Isolation System and the punch method were compared. Results: DNA extracted from FFPE tissue punches had higher DIN than that extracted from Pinpoint samples (mean [+ or -] SD, 6.18 [+ or -] 0.83 vs 5.09 [+ or -] 0.91; P < .0001), indicating less degradation. Lower DIN correlated with lower-quality metrics of NGS, that is lower percentage of unique on-target reads, average depth of coverage, and percentage of positions with coverage depth greater than or equal to 100x, 400x, and 1,000x. Conclusions: Our study demonstrated methods to harvest tissue from FFPE blocks may affect quality of DNA, which in turn has an effect on other NGS quality metrics. Key Words: Next-generation sequencing; Tissue harvesting; Formalin-fixed paraffin-embedded; DNA integrity number; Quality metrics; Preanalytical