基于单细胞靶向测序探究基因碱基突变的方法

doi:10.16288/j.yczz.20-046

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Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (7): 703-712.doi: 10.16288/j.yczz.20-046

• Technique and Method • Previous Articles

A method for reliable detection of genomic point mutations based on single-cell target-sequencing

Linan Zhao¹, Na Wang¹, Guoliang Yang², Xianbin Su¹, Zeguang Han¹()

^1. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
^2. Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China;

Received:2020-04-26 Revised:2020-06-02 Online:2020-07-20 Published:2020-06-15
Contact: Han Zeguang E-mail:hanzg@sjtu.edu.cn
Supported by:
Supported by Shanghai Jiao Tong University Scientific and Technological Innovation Funds No(2019TPA09);the National Natural Science Foundation of China No(81802806)

Abstract

Abstract:

The analysis of genomic point mutations is one of the research strategies to explore the clonal evolution of tumor cells. At present, clonal evolution of tumor cells is mainly determined by bulk sampling and sequencing of different sections of the tumor. Since this approach analyzes a mixture of different cell types, it may not accurately represent the clonal evolution of specific tumor cell populations and likely miss low frequency mutations, especially when the sequencing depths are not sufficient. To address this issue, we have developed a strategy to analyze genomic point mutations from prostate basal cell carcinoma (BCC) tissues at single-cell resolution. Firstly, we optimized the single-cell whole genome amplification procedure with HepG2 cells. Then the single cells from BCC tissue were captured by a microfluidic chip of Fluidigm and processed for whole-genome amplification. Both SCUBE3 and MST1L genomic mutations were obtained by whole exome sequencing. Finally, we examined the genomic mutations through single-cell targeted amplification and Sanger sequencing. The established method successfully reconfirmed the mutations of SCUBE3 and MST1L in BCC at single cell level. The strategy established in this study could provide a useful tool for determining the clonal evolution of tumor cells based on genomic mutations at single-cell resolution.

Key words: tumor cell, mutation, clonal evolution, single-cell

Linan Zhao, Na Wang, Guoliang Yang, Xianbin Su, Zeguang Han. A method for reliable detection of genomic point mutations based on single-cell target-sequencing[J]. Hereditas(Beijing), 2020, 42(7): 703-712.

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引物名称	引物序列(5ʹ→3ʹ)	扩增产物长度(bp)	用途
SCUBE3	F:AGGGTGTGTGTATGCGAAGG	253	碱基突变验证
SCUBE3	R:GGCCTCGCTTGTCTTGAAGT	253	碱基突变验证
MST1L	F:TTACCCGTACCTGCAGTGAG	287	碱基突变验证
MST1L	R:GACAACCTGGGAGGATGTGA	287	碱基突变验证
ACTB	F:TCTCGCAGCTCACCATGGAT	363	内参基因
ACTB	R:TGCTCGATGGGGTACTTCAG	363	内参基因

基因名称	染色体	突变位点	参考碱基	突变碱基	突变发生频率(VAF)
SCUBE3	6	35213742	C	T	0.302491103
MST1L	1	17084321	A	G	0.170385396

A method for reliable detection of genomic point mutations based on single-cell target-sequencing

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