Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Plasmids are crucial for bacterial adaptation, antibiotic resistance, and virulence.
• Low copy number of many bacterial plasmids complicates DNA extraction and sequencing.
• Accurate plasmid sequencing is essential for understanding bacterial biology and evolution.

Purpose of the Study:

• To evaluate multiple displacement amplification (MDA) for amplifying low-copy-number Salmonella plasmid DNA.
• To determine the suitability of MDA-amplified plasmid DNA for long-read sequencing and multiplexing.
• To establish an efficient protocol for closing Salmonella plasmid genomes.

Main Methods:

• Plasmid DNA isolation from nine Salmonella enterica serovars using alkaline lysis.
• Multiple displacement amplification (MDA) using Phi29 polymerase to increase DNA concentration.
• Multiplexed sequencing of amplified plasmids using Pacific Biosciences (Pacbio) Sequel (SMRT sequencing).

Main Results:

• MDA successfully generated sufficient plasmid DNA for long-read sequencing.
• All nine Salmonella plasmids (38-166 Kb) were completely closed.
• Sequencing coverage ranged from 24X to 2,582X, ensuring high accuracy.
• The protocol demonstrated effectiveness for high-molecular-weight, low-copy-number plasmids.

Conclusions:

• A combined protocol of plasmid isolation, MDA, and multiplex sequencing is effective for closing Salmonella plasmids.
• This high-throughput method significantly reduces the time and cost associated with plasmid genome closure.
• The approach facilitates comprehensive study of plasmid-borne traits like antibiotic resistance and virulence.