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

• Published in: PloS one Vol. 10; no. 9; p. e0138733
• Main Authors: Nishikawa, Yohei, Hosokawa, Masahito, Maruyama, Toru, Yamagishi, Keisuke, Mori, Tetsushi, Takeyama, Haruko
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.09.2015; Public Library of Science (PLoS)
• Subjects: Amplification; Bacteria; Bias; Bioinformatics; Chimeras; Contaminants; Contamination; Deoxyribonucleic acid; DNA; DNA polymerase; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA-directed DNA polymerase; Droplets; E coli; Earthquakes; Ecological risk assessment; Environmental assessment; Escherichia coli - cytology; Escherichia coli - genetics; Fragments; Gene sequencing; Genetic testing; Genome, Bacterial - genetics; Genomes; Genomics; Genomics - methods; Life sciences; Methods; Microfluidics; Microorganisms; Nucleic Acid Amplification Techniques - methods; Nucleotide sequence; Primers; Quality assessment; Quality control; Reagents; Reproducibility of Results; Sequence Analysis, DNA - methods; Single-Cell Analysis - methods; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0138733

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.