Abstract：Next generation sequencing (NGS) have been increasingly used in many applications. The accuracy of quantification of DNA library can directly affect the quality of the sequencing data. Thus, it is very important to develop DNA library quantification quality control method. A SYBR Green dye based digital PCR method was established to characterize the DNA library quantification reference material for Illumine NGS platform. The suitability of the proposed reference material was evaluated. The result showed that it is fit for the purpose of usage as standard curve to quantify the DNA library for illumina NGS.The results showed that the five levels of library quantitative reference materials have good linearity, and can be used as the standard curve to quantify the DNA library for Illumina NGS.
王霞,欧阳艳艳,王晶,王尚君,董莲华. 高通量测序DNA文库定量质控技术研究[J]. 计量学报, 2020, 41(10): 1308-1312.
WANG Xia,OUYANG Yan-yan,WANG Jing,WANG Shang-jun,DONG Lian-hua. Research on Characterization Method for Library DNA Reference Material for Next Generation Sequencing. Acta Metrologica Sinica, 2020, 41(10): 1308-1312.
［1］陈竺, 黄薇, 傅刚, 等. 人类基因组计划现状与展望 ［J］. 自然杂志, 2000, 22(3): 125-133.
Chen Z, Huang W, Fu G, et al. Present situation and Prospect of human genome project ［J］. Natural Journal, 2000, 22(3): 125-133.
［2］陈琛, 万海粟, 周清华. 新一代基因测序技术及其在肿瘤研究中的应用 ［J］. 中国肺癌杂志, 2010, 13(2): 154-159.
Chen C, Wan H S, Zhou Q H. New Gene Sequencing Technology and Its Application in Cancer Research［J］. Chinese Journal of Lung Cancer, 2010, 13(2): 154-159.
［3］Pfeifer G P, Besaratinia A. Mutational spectra of human cancer ［J］. Human Genetics, 2009, 125(5-6): 493-506.
［4］Moorcraft S Y, Gonzalez D, Walker B A. Understanding next generation sequencing in oncology: A guide for oncologists ［J］. Crit Rev Oncol Hematol, 2015, 96(3): 463-474.
［5］赵馨, 何天文, 尹爱华. 第二代测序技术与无创产前诊断 ［J］. 分子诊断与治疗杂志, 2014, 6(3): 198-203.
Zhao X, He T W, Yin A H. Second Generation Sequencing Technology and Noninvasive Prenatal Diagnosis ［J］. Journal of Molecular Diagnosis and Treatment, 2014, 6(3): 198-203.
［6］Wheeler D A, Srinivasan M, Egholm M, et al. The complete genome of an individual by massively parallel DNA sequencing ［J］. Nature, 2008, 452(7189): 872-876.
［7］David R B, Shankar B, Harold P S, et al. Accurate whole human genome sequencing using reversible
terminator chemistry ［J］. Nature, 2008, 456(7218): 53-59.
［8］Smith D R, Quinlan A R, Peckham H E, et al. Rapid whole-genome mutational profiling using
next generation sequencing technologies ［J］. Genome Res, 2008, 18(10): 1638-1642.
［9］Bhat S, Herrmann J, Armishaw P, et al. Single molecule detection in nanofluidic digital array enables
accurate measurement of DNA copy number ［J］. Analytical & Bioanalytical Chemistry, 2009, 394(2): 457-467.
［10］Dong L H, Meng Y, Wang J, et al. Evaluation of droplet digital PCR for characterizing plasmid reference.
material used for quantifying ammonia oxidizers and denitrifiers ［J］. Analytical and Bioanalytical Chemistry, 2014, 406(6): 1701-1712.
［11］董莲华, 孟盈, 王晶. 转Bt+CpTI基因棉花对根际土壤细菌及氨氧化细菌数量的影响 ［J］. 微生物学报, 2014, 54(3): 309-318.
Dong L H, Meng Y, Wang J. Effects of Bt+CpTI transgenic cotton on the number of bacteria and ammonia-oxidizing bacteria in rhizosphere soil ［J］. Journal of Microbiology, 2014, 54(3): 309-318.
［12］董莲华, 王晶, 傅博强, 等. 数字PCR和下一代测序方法用于KRAS基因突变检测中的可比性研究 ［J］. 计量学报, 2018, 39(3): 436-441.
Dong L H, Wang J, Fu B Q, et al. Comparability of digital PCR and next generation sequencing in the detection of KRAS gene mutations ［J］. Acta Metrologica Sinica, 2018, 39(3): 436-441.
［13］Dong L H, Meng Y, Sui Z W, et al. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material ［J］. Scientific Reports, 2015, 5: 13174.
2020, Vol. 41 
