title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells.
abstract: Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated, nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single Escherichia coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1- to 2-Mb resolution. MIDAS can potentially further the characterization of genomic diversity in many heterogeneous cell populations.
authors.affiliation: Department of Bioengineering, Institute for Genomic Medicine and Institute of Engineering in Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Gole
authors.forename: Athurva
authors.initials: A
authors.lastname: Gore
authors.forename: Andrew
authors.initials: A
authors.lastname: Richards
authors.forename: Yu-Jui
authors.initials: YJ
authors.lastname: Chiu
authors.forename: Ho-Lim
authors.initials: HL
authors.lastname: Fung
authors.forename: Diane
authors.initials: D
authors.lastname: Bushman
authors.forename: Hsin-I
authors.initials: HI
authors.lastname: Chiang
authors.forename: Jerold
authors.initials: J
authors.lastname: Chun
authors.forename: Yu-Hwa
authors.initials: YH
authors.lastname: Lo
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
mesh_terms: D002469:Cell Separation / Q000295:instrumentation*; D002874:Chromosome Mapping / Q000295:instrumentation*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase