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

Kaston Leung, Anders Klaus, Bill K. Lin, Emma Laks, Justina Biele, Daniel Lai, Ali Bashashati, Yi Fei Huang, Radhouane Aniba, Michelle Moksa, Adi Steif, Anne Marie Mes-Masson, Martin Hirst, Sohrab P. Shah, Samuel Aparicio, Carl L. Hansen

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)8484-8489
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number30
DOIs
StatePublished - Jul 26 2016

All Science Journal Classification (ASJC) codes

  • General

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