Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

Abstract

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in highthroughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform highthroughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10 −6 and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.single-cell sequencing | whole genome amplification | multiple displacement amplification | microdroplet | nanoliter volume

Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

“…By separating the genomic fragments from a single cell into multiple droplets and then performing MDA in each one, rather than confining the entire genome of a single cell in one droplet for downstream amplification, the authors were able to saturate amplification while preventing any specific sequence from dominating the process — thus improving the overall uniformity of amplification. Additionally, Leung and colleagues 62 in the Hansen laboratory presented a new technique that integrates valve- and droplet-based techniques to perform MDA in the absence of rapid flow. They controllably manipulated cells onto a substrate surface, then deposited reagent directly into the cell droplets to achieve in-drop MDA.…”

“…They controllably manipulated cells onto a substrate surface, then deposited reagent directly into the cell droplets to achieve in-drop MDA. Leung et al 62 used this approach to investigate CNVs and SNPs in single-patient-derived high-grade ovarian cancer cells. For droplets, future work will be needed to improve the overall efficiency or throughput of cell or cellular component capture, respectively.…”

Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI)

“…Multiple displacement amplification (MDA) uses a strand-displacing DNA polymerase to exponentially amplify single-stranded DNA into a hyperbranched structure. (11, 12). Multiple annealing and looping-based amplification cycles (MALBAC) employs quasi-linear amplification through looping-based amplicon protection followed by PCR (5).…”

Resource: Scalable whole genome sequencing of 40,000 single cells identifies stochastic aneuploidies, genome replication states and clonal repertoires

“…Image information acquired during cell spotting and from whole-chip fluorescence scans can be linked to single-cell libraries or used to selectively process only cells of interest. Moreover, spotting of image identified cells or nuclei more efficiently utilizes open arrays than Poisson dilution loading (Leung et al, 2016; Gao et al, 2017; Wu et al, 2015; Goldstein et al, 2017) and greatly reduces cell doublets. To aid future reimplementation of DLP+ and deployment over a wide range of cell types, we have defined the optimal working ranges of key physical and molecular reaction parameters to obtain even genome coverage without the need for genome preamplification.…”