Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

PLoS One. 2015 Sep 21;10(9):e0138733. doi: 10.1371/journal.pone.0138733. eCollection 2015.

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 microfluidic-generated 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Escherichia coli / cytology
  • Escherichia coli / genetics*
  • Genome, Bacterial / genetics*
  • Genomics / methods
  • Nucleic Acid Amplification Techniques / methods*
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods
  • Single-Cell Analysis / methods*

Substances

  • DNA, Bacterial

Grants and funding

This work is partly supported by Japan Science and Technology Agency - Core Research for Evolutional Science and Technology (JST-CREST) “Establishment of core technology for the preservation and regeneration of marine biodiversity and ecosystems”, Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) Grant-in-Aid for Scientific Basic Research (S) No. 23226010, Grant-in-Aid for Young Scientists (B) No. 26820365 and Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio-oriented Technology Research Advancement Institution).