Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Original languageEnglish
Article number5436
JournalScientific Reports
Volume7
Issue number1
DOIs
Publication statusPublished - 2017 Dec 1

Fingerprint

Microfluidics
Genome
Soil

ASJC Scopus subject areas

  • General

Cite this

@article{47e8e73065ea4f9591e88401726541f7,
title = "Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics",
abstract = "Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.",
author = "Masahito Hosokawa and Yohei Nishikawa and Masato Kogawa and Haruko Takeyama",
year = "2017",
month = "12",
day = "1",
doi = "10.1038/s41598-017-05436-4",
language = "English",
volume = "7",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",

}

TY - JOUR

T1 - Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics

AU - Hosokawa, Masahito

AU - Nishikawa, Yohei

AU - Kogawa, Masato

AU - Takeyama, Haruko

PY - 2017/12/1

Y1 - 2017/12/1

N2 - Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

AB - Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

UR - http://www.scopus.com/inward/record.url?scp=85024126714&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85024126714&partnerID=8YFLogxK

U2 - 10.1038/s41598-017-05436-4

DO - 10.1038/s41598-017-05436-4

M3 - Article

VL - 7

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 5436

ER -