title: Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification.
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.
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nishikawa
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Research Organization for Nano & Life Innovation, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Hosokawa
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yamagishi
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Mori
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Research Organization for Nano & Life Innovation, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan; Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics*; D023281:Genomics / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D004269:DNA, Bacterial