title: 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 high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput 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.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Kaston
authors.initials: K
authors.lastname: Leung
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Anders
authors.initials: A
authors.lastname: Klaus
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Bill K
authors.initials: BK
authors.lastname: Lin
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Emma
authors.initials: E
authors.lastname: Laks
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Justina
authors.initials: J
authors.lastname: Biele
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3;
authors.forename: Daniel
authors.initials: D
authors.lastname: Lai
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3;
authors.forename: Ali
authors.initials: A
authors.lastname: Bashashati
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Yi-Fei
authors.initials: YF
authors.lastname: Huang
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Radhouane
authors.initials: R
authors.lastname: Aniba
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Michelle
authors.initials: M
authors.lastname: Moksa
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;
authors.forename: Adi
authors.initials: A
authors.lastname: Steif
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Universite de Montreal, Montreal, QC, Canada H2X 0A9; Institut du Cancer de Montreal, Montreal, QC, Canada H2X 0A9; Division of Gynecologic Oncology, Department of Medicine, Universite de Montreal, Montreal, QC, Canada H3C 3J7;
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Mes-Masson
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3;
authors.forename: Martin
authors.initials: M
authors.lastname: Hirst
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3;
authors.forename: Sohrab P
authors.initials: SP
authors.lastname: Shah
authors.affiliation: Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3;
authors.forename: Samuel
authors.initials: S
authors.lastname: Aparicio
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 carl.lars.hansen@gmail.com.
authors.forename: Carl L
authors.initials: CL
authors.lastname: Hansen
mesh_terms: D000483:Alleles; D002460:Cell Line; D045744:Cell Line, Tumor; D056915:DNA Copy Number Variations; D015894:Genome, Human / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*
keywords: microdroplet; multiple displacement amplification; nanoliter volume; single-cell sequencing; whole genome amplification