Monodisperse picoliter droplets for low-bias and contamination-free reactions in single-cell whole genome amplification

Yohei Nishikawa, Masahito Hosokawa, Toru Maruyama, Keisuke Yamagishi, Tetsushi Mori, Haruko Takeyama

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidicgenerated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

Original languageEnglish
Article numbere0138733
JournalPLoS One
Volume10
Issue number9
DOIs
Publication statusPublished - 2015 Sep 21

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droplets
Amplification
Contamination
Genes
Genome
genome
cells
DNA
Microfluidics
chimerism
DNA-directed DNA polymerase
DNA-Directed DNA Polymerase
Genomics
labor
Impurities
pollution
Escherichia coli
Bacteria
genomics
Costs and Cost Analysis

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Monodisperse picoliter droplets for low-bias and contamination-free reactions in single-cell whole genome amplification. / Nishikawa, Yohei; Hosokawa, Masahito; Maruyama, Toru; Yamagishi, Keisuke; Mori, Tetsushi; Takeyama, Haruko.

In: PLoS One, Vol. 10, No. 9, e0138733, 21.09.2015.

Research output: Contribution to journalArticle

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