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.

Keywords

• cell lysis
• single-cell genomics
• genome recovery
• unculturable microbiology
• genome sequencing