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.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
He J,Du S,Tan X,Arefin A,Han CSdoi
10.2144/000114389subject
Has Abstractpub_date
2016-03-01 00:00:00pages
129-35issue
3eissn
0736-6205issn
1940-9818pii
000114389journal_volume
60pub_type
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