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Main Title: The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms
Author(s): Liu, Yuguang
Schulze-Makuch, Dirk
De Vera, Jean-Pierre
Cockell, Charles
Leya, Thomas
Baqué, Mickael
Walther-Antonio, Marina
Type: Article
Language Code: en
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.
Issue Date: 25-Jul-2018
Date Available: 29-Aug-2019
DDC Class: 570 Biowissenschaften; Biologie
Subject(s): bacteria lysis protocol
microalgae lysis
single-cell multiple displacement amplification
Sponsor/Funder: EC/FP7/339231/Habitability of Martian Environments: Exploring the Physiological and Environmental Limits of Life/HOME
Journal Title: Micromachines
Publisher: MDPI
Publisher Place: Basel
Volume: 9
Issue: 8
Article Number: 367
Publisher DOI: 10.3390/mi9080367
EISSN: 2072-666X
Appears in Collections:Zentrum für Astronomie und Astrophysik » Publications

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