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 Baque, Marina Walther-Antonio*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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
Article number367
Number of pages17
JournalMicromachines
Volume9
Issue number8
DOIs
Publication statusPublished - 25 Jul 2018

Keywords

  • bacteria lysis protocol
  • microalgae lysis
  • single-cell multiple displacement amplification
  • MULTIPLE DISPLACEMENT AMPLIFICATION
  • HEAT-SHOCK
  • DNA
  • IMPACT
  • FUTURE
  • WALL
  • CORYNEBACTERIUM
  • CYANOBACTERIUM
  • DITHIOTHREITOL
  • PEPTIDOGLYCAN

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