Centrifugal-Driven Droplet Generation Method with Minimal Waste for Single-Cell Whole Genome Amplification

Abstract

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.

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

“…We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units. In contrast to the state of the art, where until now only one-plex ddPCR assays have been automated by centrifugal microfluidics [9,[17][18][19], we present an increased multiplexing degree of four. This elevated degree of multiplexing is particularly relevant for cancer diagnostics, where a comprehensive overview of the mutation status of a patient must be generated from a limited sample amount [3].…”

“…In the recent past, centrifugal microfluidics developed into a mature technology which enabled the efficient miniaturization, parallelization and integration of assays for both sample preparation and analysis [13][14][15]. In the past, several groups have presented centrifugal, microfluidic cartridges that were able to emulsify a reaction mix [6,9,[16][17][18][19][20]. We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units.…”

Off‐Chip Vertical Step Emulsification Droplets Preparation Device Applied for Droplet Digital PCR

“…Most of the previous work focused on the effect of the opening angle of nozzles, [ 31 ] the viscosity of oil phase, [ 36 ] and the nozzle geometry [ 29 ] to the uniformity of droplets. However, VSE device is an off‐chip microfluidic system, which can adjust the droplet size by adjusting the inner diameter of the PFA capillary, the rotating angle of positioning screw, and the speed ( V value) of mechanical pump, respectively.…”

“…[5] The critical step of dPCR is to separate the independent microreaction chambers with equal and stable in structure. However, the on-chip ddPCR device still limits the droplets throughput because of the fixed step height of the microchip and the width of nozzles, the droplets throughput has not been greatly improved, such as EDGE devices designed by Sahin and Schroën, [28] centrifugal-driven droplet generation method proposed by Li et al, [29] and the millipede device designed by Amstad et al [30] But the off-chip ddPCR device removes this limitation and greatly improves the droplet throughput of the ddPCR device. For instance, the cross-interface emulsification method proposed by Xu et al, [31] the centrifuge-based step emulsification device by Shin et al, [32] and the off-chip monodisperse droplet generation method by Chen et al [33] Although their methods broke the limitation of low droplet throughput, the cost of their droplet preparation system is higher, and the manufacturing process is more difficult, which brings some difficulties to the inexperienced researchers in the field of system design and manufacturing.…”

“…Compared with the T‐junctions method and flow focusing method, the step emulsification method is simple in operation, high in throughput, and stable in structure. However, the on‐chip ddPCR device still limits the droplets throughput because of the fixed step height of the microchip and the width of nozzles, the droplets throughput has not been greatly improved, such as EDGE devices designed by Sahin and Schroën, [ 28 ] centrifugal‐driven droplet generation method proposed by Li et al., [ 29 ] and the millipede device designed by Amstad et al. [ 30 ] But the off‐chip ddPCR device removes this limitation and greatly improves the droplet throughput of the ddPCR device.…”