Improved Lysis of Single Bacterial Cells by a Modified Alkaline-Thermal Shock Procedure

Abstract

Single-cell genomics (SCG) is a recently developed tool to study the genomes of unculturable bacterial species. SCG relies on multiple-strand displacement amplification (MDA), PCR, and next-generation sequencing (NGS); however, obtaining sufficient amounts of high-quality DNA from samples is a major challenge when performing this technique. Here we present an improved bacterial cell lysing procedure that combines incubation in an alkaline buffer with a thermal shock (freezing/heating) treatment to yield highly intact genomic DNA with high efficiency. This procedure is more efficient in lysing Bacillus subtilis and Synechocystis cells compared with two other frequently used lysis methods. Furthermore, 16S ribosomal RNA gene and overall genome recovery were found to be improved by this method using single cells from a Utah desert soil community or Escherichia coli single cells, respectively. The efficiency of genome recovery for E. coli single cells using our procedure is comparable with that of the REPLI-g Single Cell (sc) Kit, but our method is much more economical. By providing high-quality genome templates suitable for downstream applications, our procedure will be a promising improvement for SCG research.

The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

“…Chemicals including phenol and spermine have been proven effective for degrading bacterial cell walls, however, due to the toxicity, a fume hood is required, thus, making it difficult to use in microfluidic systems. Freeze-thaw combined with alkaline treatment has been proven highly efficient for the lysis of bacterial single cells for SC-WGA in well plates [ 43 ], but requires placing the plates in −80 °C freezer for 1 h, therefore it is not applicable for microfluidic experimental setups with complex controls.…”

“…Therefore, in this work, we develop a bacterial single-cell lysis protocol as a guideline for MDA-based bacterial SC-WGA in microfluidic platforms that produces >25 ng of genomic DNA per cell, sufficient for downstream library preparation. This on-chip protocol combines three primary bacterial lysis methods which include thermal [ 15 , 43 , 44 ], enzymatic [ 45 , 46 ] and chemical lysis [ 47 , 48 , 49 ] and was tested on both gram-positive and gram-negative bacterial species for subsequent on-chip SC-WGA. In this study, Corynebacterium glutamicum was used as a typical gram-positive model whose cell wall is thicker than most gram-negative species.…”

Microbial single-cell omics: the crux of the matter

“…i After quality assessment, trimming, and/or normalization of the sequencing reads, bioinformatics tools can conduct the assembly, ORF calling, and annotation of the genes, as well as pathway reconstruction and gene comparisons. Created with BioRender method; however, more efficient lysis of cells from complex communities can be accomplished by using a combination of different lysis methods (i.e., physical-like freeze-thaw cycles, chemical, or enzymatic) (Hall et al 2013;He et al 2016;Stepanauskas et al 2017).…”

Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

“…Moreover, rRNA and tRNA molecules usually represent over 90% of total RNA, but offer limited biological information and should be excluded in the amplification process, as most researches focus on mRNA and other rare molecules. With a complicated cell wall, harsher conditions are typically required to lyze a microbial cell, which may lead to damage or loss of RNA, and accuracy and efficiency of the downstream transcriptomic analysis ( Khan and Yadav, 2004 ; Hall et al, 2013 ; Heera et al, 2015 ; He et al, 2016 ). More importantly, unlike genomic analysis, in which the methods for mammalian cells could be also readily applied to prokaryotic microbes, not all methods for mammalian single-cell transcriptomic analysis could be used to microbes.…”