Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification.
Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10(5)) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.
Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing 100871, China;
Yusi
Y
Fu
Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing 100871, China;
Chunmei
C
Li
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138;
Sijia
S
Lu
Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing 100871, China;
Wenxiong
W
Zhou
Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China;
Fuchou
F
Tang
Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing 100871, China; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; xie@chemistry.harvard.edu yanyi@pku.edu.cn.
X Sunney
XS
Xie
Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; College of Engineering, Peking University, Beijing 100871, China xie@chemistry.harvard.edu yanyi@pku.edu.cn.
Yanyi
Y
Huang
D056915:DNA Copy Number Variations / Q000235:genetics; D004171:Diploidy; D004655:Emulsions / Q000737:chemistry*; D059472:Exome / Q000235:genetics; D015894:Genome, Human; D019073:HT29 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
D004655:Emulsions
emulsion; microfluidics; sequencing; single cell; whole-genome amplification