黑色素瘤福尔马林固定石蜡包埋（FFPE）样本用于二代测序（NGS）文库的适用性：下游分子检测的时间和质量阈值

Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests.

作者信息

Millán-Esteban David, Reyes-García Diana, García-Casado Zaida, Bañuls José, López-Guerrero José Antonio, Requena Celia, Rodríguez-Hernández Aranzazu, Traves Víctor, Nagore Eduardo

机构信息

Laboratorio de Biología Molecular, Instituto Valenciano de Oncología, Valencia, Spain.

Universidad Católica de Valencia San Vicente Mártir, Valencia, Spain.

出版信息

Biotechniques. 2018 Aug;65(2):79-85. doi: 10.2144/btn-2018-0016.

DOI:10.2144/btn-2018-0016

PMID:30091391

Abstract

The use of NGS in clinical practice for precision diagnosis requires a quality starting material. Despite the broadly established use of formalin-fixed paraffin-embedded (FFPE) samples in molecular testing, these usually have low-quality DNA. We established a method to determine the suitability of melanoma FFPE samples for an amplicon-based NGS custom panel analysis. DNA was extracted from unstained melanoma samples and wide local excision samples. Amplicon-based libraries were constructed and tested using time and quality parameters as variables. Time elapsed from sample retrieval >7 years, a quality control value > 5.63 and a DNA integrity value < 2.05 indicated samples were not suitable. A decision tree is provided with rate of samples suitable for analysis according to the combination of these parameters.

摘要

在临床实践中使用二代测序（NGS）进行精准诊断需要高质量的起始材料。尽管福尔马林固定石蜡包埋（FFPE）样本在分子检测中已被广泛应用，但这些样本的DNA质量通常较低。我们建立了一种方法来确定黑色素瘤FFPE样本是否适合基于扩增子的NGS定制 panel 分析。从未染色的黑色素瘤样本和广泛局部切除样本中提取DNA。构建基于扩增子的文库，并将时间和质量参数作为变量进行测试。样本取回后经过的时间>7年、质量控制值>5.63以及DNA完整性值<2.05表明样本不适合。根据这些参数的组合提供了一个决策树，显示适合分析的样本比例。