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 plat...

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Autores principales:	Masahito Hosokawa, Yohei Nishikawa, Masato Kogawa, Haruko Takeyama
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4530380e612d483d99e0d4b9a6b29f66
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spelling	oai:doaj.org-article:4530380e612d483d99e0d4b9a6b29f662021-12-02T12:32:56ZMassively parallel whole genome amplification for single-cell sequencing using droplet microfluidics10.1038/s41598-017-05436-42045-2322https://doaj.org/article/4530380e612d483d99e0d4b9a6b29f662017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-05436-4https://doaj.org/toc/2045-2322Abstract 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.Masahito HosokawaYohei NishikawaMasato KogawaHaruko TakeyamaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Masahito Hosokawa Yohei Nishikawa Masato Kogawa Haruko Takeyama Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
description	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.
format	article
author	Masahito Hosokawa Yohei Nishikawa Masato Kogawa Haruko Takeyama
author_facet	Masahito Hosokawa Yohei Nishikawa Masato Kogawa Haruko Takeyama
author_sort	Masahito Hosokawa
title	Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
title_short	Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
title_full	Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
title_fullStr	Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
title_full_unstemmed	Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
title_sort	massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/4530380e612d483d99e0d4b9a6b29f66
work_keys_str_mv	AT masahitohosokawa massivelyparallelwholegenomeamplificationforsinglecellsequencingusingdropletmicrofluidics AT yoheinishikawa massivelyparallelwholegenomeamplificationforsinglecellsequencingusingdropletmicrofluidics AT masatokogawa massivelyparallelwholegenomeamplificationforsinglecellsequencingusingdropletmicrofluidics AT harukotakeyama massivelyparallelwholegenomeamplificationforsinglecellsequencingusingdropletmicrofluidics
_version_	1718393913401671680

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

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