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 (10⁵) 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.Single-cell sequencing, characterization the genome of individual cells, is highly needed for studying scarce and/or precious cells, which are inaccessible for conventional bulk genome characterization, and for probing genomic variations of a heterogeneous population of cells (1–3). Recently single-cell genomics has unveiled unprecedented details of various biological processes, such as tumor evolution (4–6), embryonic development (7), and neural somatic mosaicism (8). Single-cell whole-genome amplification (WGA) is required to generate enough replicates of genomic DNAs for library preparation in conjunction with current sequencing protocols. Single-cell WGA has been increasingly used in cutting-edge clinical diagnostic applications such as molecular subtyping of single tumor cells (4, 9) and preimplantation genetic screening of in vitro fertilized embryos (10).An ideal single-cell WGA method should have high uniformity and accuracy across the whole genome. The WGA uniformity is critical for copy number variation (CNV) detection, whereas the WGA accuracy is essential for avoiding single-nucleotide variation (SNV) detection errors, either false positives or false negatives. The false positives arise from misincorporation of wrong bases in the first few cycles of WGA. In a diploid human cell, the false negatives primarily arise from the allelic dropout (ADO), i.e., heterozygous mutations are mistaken as homozygous ones because of the lack of amplification in one of the two alleles (11).Existing WGA chemistry includes degenerate oligonucleotide-primed PCR (DOP-PCR) (12), multiple displacement amplification (MDA) (13–17), and multiple annealing and looping-based amplification cycles (MALBAC) (4, 18, 19), which have successively achieved genome analysis at the single-cell level. DOP-PCR is based on PCR amplification of the fragments flanked by universal priming sites, and provides high accuracy for detecting CNVs in single cells but has low coverage and high false-positive and false-negative rates for calling SNVs (5). MDA has a much improved coverage but tends to have lower precision/sensitivity in CNV determination due to its variation of the amplification gain along the genome, not reproducible from cell to cell (20). By virtue of quasilinear amplification, MALBAC suppresses the random bias of amplification and exhibits reduced ADO rates, yielding low false negatives for SNV detection (2, 11, 18, 19). Notwithstanding its drawbacks, MDA still offers comparable or higher genome coverage than MALBAC, at least for single diploid cells, possibly taking advantage of the randomness (2). In fact, even higher coverage has been obtained for cells with aneuploidy, such as dividing cells (21), and cancer cells (22). MDA’s main advantage is its lower false-positive rate for SNV detection on account of the use of Phi-29, a highly processive polymerase with high fidelity.Microfluidic devices have been carried out for single-cell WGA (16, 20, 23, 24), allowing avoidance of contaminations and high-throughput analyses of multiple single cells in parallel. The small total reaction volumes (microliters to nanoliters- or picoliters) of the microfluidic devices not only facilitate the efficiency of reactions but also allow significant cost reduction for enzymes and regents used. It was reported that the nanoliter volume of a microfluidic device improved uniformity of the amplification compared with microliter devices in the WGA of single bacterial cells (20).Here, we report a method, emulsion whole-genome amplification (eWGA), to use the small volume of aqueous droplets in oil to better the WGA chemistry for uniform amplification of a single cell’s genome. By distributing single-cell genomic DNA fragments into a large number (10⁵) of picoliter droplets, a few DNA fragments in each droplet is allowed to reach saturation of DNA amplification. After merging the droplets by demulsification, the differences in amplification gain among the DNA fragments are significantly minimized.Although this approach can be used for any chemistry of WGA, we take MDA as an example to greatly reduce the random bias of amplification by separating the reactions into a large amount of emulsion droplets. We carried out detailed comparison with MDA, MALBAC, and DOP-PCR performed in tube using single cells from normal diploid human cells and a monoclonal human cancer cell line with inherited CNVs. Our results indicate that eWGA not only offers higher coverage but also enables simultaneous detection of SNVs and CNVs with higher accuracy and finer resolution, outperforming the prevailing single-cell amplification methods in many aspects.