组织块打孔与未染色切片上的针尖式提取对下一代测序质量指标的影响：提高 DNA 完整性。

Effects of Improved DNA Integrity by Punch From Tissue Blocks as Compared to Pinpoint Extraction From Unstained Slides on Next-Generation Sequencing Quality Metrics.

机构信息

University of Alabama at Birmingham.

出版信息

Am J Clin Pathol. 2019 Jun 5;152(1):27-35. doi: 10.1093/ajcp/aqz014.

DOI:10.1093/ajcp/aqz014

PMID:30892602

Abstract

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 ± SD, 6.18 ± 0.83 vs 5.09 ± 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 100×, 400×, and 1,000×.

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.

摘要

目的

比较两种福尔马林固定石蜡包埋（FFPE）组织采集方法对 DNA 质量和下一代测序（NGS）质量指标的影响。

方法

比较了通过 pinpoint Slide DNA 分离系统和打孔法采集的 199 个连续样本的 DNA 提取和测序产生的 DNA 完整性数（DIN）和 NGS 质量指标。

结果

从 FFPE 组织打孔中提取的 DNA 的 DIN 高于 pinpoint 样本（平均值±标准差，6.18±0.83 对 5.09±0.91；P＜0.0001），表明降解程度较低。较低的 DIN 与 NGS 的较低质量指标相关，即较低的独特靶向读取百分比、平均覆盖深度以及覆盖深度大于或等于 100×、400×和 1000×的位置百分比。