Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.
Single-cell RNA sequencing (scRNA-seq) is a powerful method in investigating single-cell heterogeneity to reveal rare cells, identify cell subpopulations, and construct a cell atlas. Conventional benchtop methods for scRNA-seq, including multistep operations, are labor intensive, reaction inefficient, contamination prone, and reagent consuming. Here we report a digital microfluidics-based single-cell RNA sequencing (digital-RNA-seq) for simple, efficient, and low-cost single-cell mRNA measurements. Digital-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of scRNA-seq. To overcome the current problems of single-cell isolation in efficiency, integrity, selectivity, and flexibility, we propose a new strategy, passive dispensing method, relying on well-designed hydrophilic–hydrophobic microfeatures to rapidly generate single-cell subdroplets when a droplet of cell suspension is encountered. For sufficient cDNA generation and amplification, digital-RNA-seq uses nanoliter reaction volumes and hydrophobic reaction interfaces, achieving high sensitivity in gene detection. Additionally, the stable droplet handling and oil-closed reaction space featured in digital-RNA-seq ensure highly accurate measurement. We demonstrate the functionality of digital-RNA-seq by quantifying heterogeneity among single cells, where digital-RNA-seq shows excellent performance in rare transcript detection, cell type differentiation, and essential gene identification. With the advantages of automation, sensitivity, and accuracy, digital-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.
High-quality whole-genome amplification (WGA) of individual cells is the primary step for characterizing the genetic information on single cells in biology and medicine. As the most popular single-cell WGA method, multiple displacement amplification (MDA) is often plagued by the nonuniform amplification. The droplet MDA has been an innovative tool to solve this dilemma by mitigating the amplification bias and increasing the genomic coverage. Despite these advantages, the time-consuming droplet generation process, the waste of small volume samples and the difficulty of parallel operation for multiple single-cell samples remain major obstacles. Herein, we introduce a centrifugal-driven droplet generation method for rapid and convenient generation of uniform droplets from a relatively small volume sample (5 μL) in 60s with more than 98% sample utilization. We have performed quantitative digital droplet PCR using this method, demonstrating its capability of amplifying nucleic acids at the single-molecule level. Single-cell centrifugal-driven droplet MDA (cd-MDA) has also been conducted for single-cell sequencing, achieving uniform amplification and broad genomic coverage. With the single-molecule sensitivity, minimum sample waste, high genomic coverage, and excellent sequencing evenness, this centrifugal-driven droplet generation method is promising for convenient and scalable use in digital PCR and single-cell whole-genome research.
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