P2-127 A Novel Method to Achieve Complete Low-copy Number Plasmid Sequences of Salmonella enterica

Tuesday, July 11, 2017
Exhibit Hall (Tampa Convention Center)
Kuan Yao , U.S. Food and Drug Administration , College Park , MD
Narjol Gonzalez-Escalona , U.S. Food and Drug Administration , College Park , MD
Julien Marquis , Nestlé Institute of Health Sciences , Lausanne , Switzerland
Marc Allard , U.S. Food and Drug Administration , College Park , MD
Maria Hoffmann , U.S. Food and Drug Administration , College Park , MD
Introduction: Plasmids play a major role in bacterial adaption to environmental stress and often contribute to antibiotic resistance and disease virulence. To study plasmid biology the complete plasmid sequence is essential. The majorities of antibiotic resistance and virulence plasmids in Salmonella have a low copy number and unfortunately, are hard to extract and to sequence. The existent protocol consists in a laborious transformation of the plasmid solution, extracted from Salmonella, into Escherichia coli in order to achieve enough DNA for accurate sequencing. Therefore there is a need for a fast method to produce high quantity and good quality of plasmid DNA.

Purpose: The purpose of this study was to test the efficiency of rolling circle amplification for replicating Salmonella plasmid DNA to a satisfactory amount making it suitable for sequencing.

Methods: Six Salmonella enterica isolates, carrying known low copy number plasmids, representing five different serovars were cultured and the plasmids were isolated using the Qiagen Plasmid Mini Kit. The plasmid DNA solution was amplified using the REPLI-g Mitochondrial DNA kit. The amplified plasmids were sequenced on the Pacific Biosciences RSII sequencer.

Results: High copies of Salmonella plasmid DNA were acquired through rolling circle amplification for sequencing. We were able to completely close all five Salmonella plasmids (size ranged from 38 Kb to 190 Kb) with a sequencing coverage from 2,400X to 5,800X. All de novo assembled plasmids aligned 100% with their reference genome on NCBI.

Significance: This novel protocol, consisting of plasmid isolation, replication and sequencing, is a valuable tool for closing high-molecular weight and low-copy-number plasmids. The rolling circle amplification was proven to be an effective and fast method for plasmid DNA replication by passing the low copy number hindrance without laborious transformation into Escherichia coli. This protocol will be beneficial for high throughout plasmid sequencing.