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

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Understanding genomic heterogeneity in complex systems requires sequencing single cells.
• Whole genome amplification (WGA) is crucial but often suffers from nonuniformity, impacting accuracy.
• Existing high-throughput single-cell WGA methods lack robust performance.

Purpose of the Study:

• To introduce droplet multiple displacement amplification (MDA) for high-throughput single-cell WGA.
• To evaluate the performance of droplet MDA in terms of amplification uniformity, genome coverage, and robustness.
• To demonstrate the utility of droplet MDA for detecting single-nucleotide variants (SNVs) and copy number variants (CNVs) in single cells.

Main Methods:

• Developed droplet multiple displacement amplification (MDA) using commercial liquid dispensing in nanoliter volumes.
• Characterized droplet MDA performance using a dataset of 129 normal diploid cells.
• Assessed SNV detection via targeted and whole genome sequencing, and CNV detection in cancer cell lines and patient samples.

Main Results:

• Droplet MDA demonstrated superior amplification uniformity, genome coverage, and robustness compared to previous methods.
• Achieved up to 80% genome coverage at 5× sequencing depth with a median allelic dropout of 15% for SNV detection.
• Enabled detection of CNVs as small as 30 kb in single cells and 9 Mb in clinical samples.

Conclusions:

• Droplet MDA offers an accessible, scalable, and accurate method for high-throughput single-cell WGA.
• The method facilitates robust SNV and CNV measurements across large numbers of single cells.
• This advancement supports deeper understanding of genomic heterogeneity in complex biological systems.