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.
Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.
Biopanning, a common affinity selection approach in phage display, has evolved numerous ligands for diagnosis, imaging, delivery, and therapy applications. However, traditional biopanning has suffered from time-consuming processes, highly-repetitive procedures...
Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on
d
igital microfluidics for
d
igital counting of
s
ingle-
c
ell
C
opy
N
umber
V
ariation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.
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