The development of an effective bacterial single-cell lysis method suitable for whole genome amplification in microfluidic platforms

Yuguang Liu, Dirk Schulze-Makuch, Jean Pierre de Vera, Charles Cockell, Thomas Leya, Mickael Baqué, Marina Walther-Antonio

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to > 5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

Original languageEnglish (US)
Article number367
JournalMicromachines
Volume9
Issue number8
DOIs
StatePublished - Jul 25 2018

Fingerprint

Microfluidics
Amplification
Genes
DNA
Cells
Fluids

Keywords

  • Bacteria lysis protocol
  • Microalgae lysis
  • Single-cell multiple displacement amplification

ASJC Scopus subject areas

  • Control and Systems Engineering
  • Mechanical Engineering
  • Electrical and Electronic Engineering

Cite this

The development of an effective bacterial single-cell lysis method suitable for whole genome amplification in microfluidic platforms. / Liu, Yuguang; Schulze-Makuch, Dirk; de Vera, Jean Pierre; Cockell, Charles; Leya, Thomas; Baqué, Mickael; Walther-Antonio, Marina.

In: Micromachines, Vol. 9, No. 8, 367, 25.07.2018.

Research output: Contribution to journalArticle

Liu, Yuguang ; Schulze-Makuch, Dirk ; de Vera, Jean Pierre ; Cockell, Charles ; Leya, Thomas ; Baqué, Mickael ; Walther-Antonio, Marina. / The development of an effective bacterial single-cell lysis method suitable for whole genome amplification in microfluidic platforms. In: Micromachines. 2018 ; Vol. 9, No. 8.
@article{246596b0800544979600004a72994b65,
title = "The development of an effective bacterial single-cell lysis method suitable for whole genome amplification in microfluidic platforms",
abstract = "Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80{\%} of single-cell replicates that lead to > 5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100{\%} success rate.",
keywords = "Bacteria lysis protocol, Microalgae lysis, Single-cell multiple displacement amplification",
author = "Yuguang Liu and Dirk Schulze-Makuch and {de Vera}, {Jean Pierre} and Charles Cockell and Thomas Leya and Mickael Baqu{\'e} and Marina Walther-Antonio",
year = "2018",
month = "7",
day = "25",
doi = "10.3390/mi9080367",
language = "English (US)",
volume = "9",
journal = "Micromachines",
issn = "2072-666X",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "8",

}

TY - JOUR

T1 - The development of an effective bacterial single-cell lysis method suitable for whole genome amplification in microfluidic platforms

AU - Liu, Yuguang

AU - Schulze-Makuch, Dirk

AU - de Vera, Jean Pierre

AU - Cockell, Charles

AU - Leya, Thomas

AU - Baqué, Mickael

AU - Walther-Antonio, Marina

PY - 2018/7/25

Y1 - 2018/7/25

N2 - Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to > 5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

AB - Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to > 5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

KW - Bacteria lysis protocol

KW - Microalgae lysis

KW - Single-cell multiple displacement amplification

UR - http://www.scopus.com/inward/record.url?scp=85054931641&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054931641&partnerID=8YFLogxK

U2 - 10.3390/mi9080367

DO - 10.3390/mi9080367

M3 - Article

AN - SCOPUS:85054931641

VL - 9

JO - Micromachines

JF - Micromachines

SN - 2072-666X

IS - 8

M1 - 367

ER -