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中华临床实验室管理电子杂志 ›› 2020, Vol. 08 ›› Issue (02) : 105 -112. doi: 10.3877/cma.j.issn.2095-5820.2020.02.008

所属专题: 文献

自动化与信息化

福尔马林固定石蜡包埋样本全基因组文库构建流程优化
欧小华1, 刘菲菲2, 毛琳琳2, 周丹燕1, 胡昌明1, 赵薇薇2,()   
  1. 1. 510005 广州,广州金域医学检验中心有限公司临床基因组检测中心
    2. 510005 广州,广州金域医学检验中心有限公司临床基因组检测中心;510005 广州,广州金域医学检验集团股份有限公司
  • 收稿日期:2019-07-22 出版日期:2020-05-28
  • 通信作者: 赵薇薇
  • 基金资助:
    广州市科技计划项目(201802020030); 广州市产业领军人才集聚工程(CXLJTD-201603)

Optimization of whole genome library construction process for formalin-fixed and paraffin-embedded tissue

Xiaohua Ou1, Feifei Liu2, Linlin Mao2, Danyan Zhou1, Changming Hu1, Weiwei Zhao2,()   

  1. 1. Clinical Genome Center, KingMed Center for Clinical Laboratory Co., Ltd
    2. Clinical Genome Center, KingMed Center for Clinical Laboratory Co., Ltd; Guangzhou KingMed Diagnostics Group Co., Ltd. Guangzhou 510005, China
  • Received:2019-07-22 Published:2020-05-28
  • Corresponding author: Weiwei Zhao
  • About author:
    Corresponding author: Zhao Weiwei, Email:
引用本文:

欧小华, 刘菲菲, 毛琳琳, 周丹燕, 胡昌明, 赵薇薇. 福尔马林固定石蜡包埋样本全基因组文库构建流程优化[J]. 中华临床实验室管理电子杂志, 2020, 08(02): 105-112.

Xiaohua Ou, Feifei Liu, Linlin Mao, Danyan Zhou, Changming Hu, Weiwei Zhao. Optimization of whole genome library construction process for formalin-fixed and paraffin-embedded tissue[J]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2020, 08(02): 105-112.

目的

优化建立适合本实验室针对人源福尔马林固定石蜡包埋(Formalin-Fixed and Paraffin-Embedded, FFPE)样本的全基因组(Genomic DNA, gDNA)文库构建方案。

方法

探讨并设计了在FFPE全基因组文库构建各环节优化条件,分析比较FFPE DNA文库片段大小及文库转化率,以选择最优的文库构建流程。

结果

最终确定DNA起始量50 ng、20 min片段化酶切时间,采用4×稀释的接头进行20 min连接,经过2次特定比例的磁珠筛选方案进行文库构建,实现片段主峰在300 bp~400 bp左右,文库转化率达60%。

结论

本操作流程针对FFPE样本可以保证高质量的文库构建,同时实现较高文库转化效率。

Objective

To optimize the establishment of a whole genome (Genomic DNA, gDNA) library construction scheme suitable for human formalin-fixed and paraffin embedded (FFPE) tissue.

Methods

The optimization conditions for the FFPE whole-genome library construction were discussed and designed, and the FFPE DNA library fragment size and library conversion rate were analyzed and compared to select the optimal library construction process.

Results

DNA input was not less than 50 ng, enzyme digestion time was 20 min, 4× dilution adapter was ligated 20 min, fragment size selection by beads were 0.8× and then 1×, Fragment peak could be controlled about 300 bp~400 bp, library conversion rate was up to 60%.

Conclusion

The operating procedure established in this project ensures high quality library construction and high library conversion efficiency for FFPE samples.

表1 检测用试剂清单
表2 各步骤所需仪器清单
图1 文库构建流程示意图
图2 测试优化方案图
表3 稳定性和重复性评估设计
表3 片段筛选方案优化数据
表4 接头浓度优化数据
表5 连接时间优化数据
表6 稳定性和重复性数据
图3 DNA浓度(质量)反映文库主峰及转化率关系
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