Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

Abstract

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.

Keywords: emulsion; microfluidics; sequencing; single cell; whole-genome amplification.

Fig. 1.

The experimental process of eWGA-seq and emulsion generation. (A) A single cell is lysed and then mixed with MDA reaction buffer in a tube. The solution was either directly used for conventional MDA, generating unevenly amplified DNA fragments, or used for emulsion generation in a microfluidics cross-junction device, resulting in uniformly distributed aqueous reaction droplets and evenly amplified DNA fragments. (B) The microfluidics cross-junction. Reaction buffer and mineral oil are driven by compressed air with proper pressure to achieve uniform water-in-oil emulsion. The cross-section of the channel is 105 × 100 μm. The speed of emulsion generation is ∼35,000 per min. (Scale bar: 300 μm.) (C) All droplets are collected into a 200-μL microcentrifuge tube and incubated at 30 °C to perform eWGA. (D) The emulsion is stable during the reaction. (Scale bar: 100 μm.)

Fig. 2.

The comparison of WGA methods for sequencing single HUVECs. (A) The copy number across the whole genome with a mean bin size of 52.4 kb; black line shows the expected value. (B) The density histogram of copy number distribution (bin size, 502 kb). (C) The Lorenz curves of coverage uniformity for single cells amplified by eMDA, MALBAC, conventional MDA, and unamplified genomic DNA. (D) The power spectrum of read density as a function of spatial frequency. (E) Copying-error rate of single-cell WGA methods. (F) ADO rate of single-cell WGA methods. (G) The ratio of the sequencing read originated from major pollutes in single-cell eMDA and conventional MDA experiments.

Fig. 3.

The comparison of WGA methods for sequencing single HT-29 cells. (A) The circos plot (27) showing the copy number profiles from unamplified genomic DNA and from a single cell amplified by eMDA. (B) The zoomed-in copy number distribution of chr3 and chrX with a binning size of 52.4 kb. The smallest CNV detected is 5 bins. (C) Heat map showing copy number gains and losses of single cells with different amplification methods, with unamplified genomic DNA as reference. The correlation efficiencies between single-cell WGA methods and bulk reference are also listed. (D) The CNV detection sensitivity under different bin size threshold of single-cell WGA methods. The filled area represents the SD of each method. (E) The coverage ratio of exome captured single-cell WGA samples using unamplified sample as reference. (F) The homozygous SNVs detected in single cells using different WGA methods. The blue line shows the number of homozygous SNVs identified in the unamplified sample. The blue bars show the SNVs that matched bulk reference, whereas the red bars show the discordant SNVs.