Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

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...

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Published inNature biotechnology Vol. 31; no. 12; pp. 1126 - 1132
Main Authors Gole, Jeff, Gore, Athurva, Richards, Andrew, Chiu, Yu-Jui, Fung, Ho-Lim, Bushman, Diane, Chiang, Hsin-I, Chun, Jerold, Lo, Yu-Hwa, Zhang, Kun
Format Journal Article
LanguageEnglish
Published United States Nature Publishing Group 01.12.2013
Subjects
Cell Separation - instrumentation
Cells
Chromosome Mapping - instrumentation
Cloning
Cloning, Molecular - methods
Deoxyribonucleic acid
DNA
DNA - genetics
DNA polymerases
DNA sequencing
DNA-Directed DNA Polymerase - genetics
E coli
Equipment Design
Equipment Failure Analysis
Gene amplification
Genetic aspects
Genomics
High-Throughput Nucleotide Sequencing - instrumentation
High-Throughput Nucleotide Sequencing - methods
Mammals
Methods
Microorganisms
Mutation
Nanotechnology - instrumentation
Nanotechnology - methods
Nucleotide sequencing
Physiological aspects
Reactors
United States
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Summary: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.
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Present address: Department of Animal Science, National Chung Hsing University, Taichung, Taiwan
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt.2720