Plasmid Detection and Complete Plasmid DNA Sequencing

Bacterial plasmids

Bacterial plasmids are circular or linear double-stranded DNA molecules defined by their capability of autonomous replication in the hosts. They are critical sources for microbial evolution and genome innovation due to their ability to acquire foreign DNA sequences and transfer among bacteria and between distantly related organisms, like transferring from bacteria to eukaryotes via conjugation and mobilization. Bacterial plasmids undergo a higher frequency of genetic recombination than chromosomes. Plasmid genome, also known as the accessory or flexible genome, does not encode basic survival functions, but instead, it contributes to exploiting particular environmental niches, pathogenicity, degradation of aromatics, and antimicrobial resistance (AMR). Resistance to antibiotics has directly and sharply increased the number of multidrug-resistant bacteria. Thus, considerable effort has been made for plasmid detection and monitoring by using methods like complete plasmid DNA sequencing.

Figure 1. Autonomous replication and genomic integration of bacterial plasmids.

Methods for plasmid detection

There are several approaches for plasmid detection, based on PCR, gel electrophoresis, optical mapping or sequencing.

1. PCR-based replicon typing. This approach targets the conserved replicon sites of plasmid, and multiplex PCR can be expanded to simultaneously target many replicons. Although cheap and fast, this approach is hard to cover all novel plasmid groups.

2. Pulsed field gel electrophoresis (PFGE). This method separates digested DNA on a gel matrix by applying an electric field that periodically changes direction. However, it typically takes several days to reveal the size and number of plasmids in an isolate.

   Figure 2. Pulsed field gel electrophoresis (Hu & Manos 2015).

3. Optical mapping of plasmids. This method relying on the stretching of plasmid DNA can be used to depict the sequence of a plasmid. But optical mapping may not be suitable for the detection of short (< 50 Kb) plasmids.

   Figure 3. Optical mapping of plasmids (Bogas et al., 2017).

4. Sequencing-based plasmid detection. With the development of high-throughput sequencing technologies, it is possible to obtain all the information contained in plasmids. Whole genome sequencing (WGS) can capture both bacterial and plasmid sequence data. Using algorithms such as PlasmidSeeker and plasmidSPAdes can extract and assemble plasmid data from WGS projects. However, these tools can only detect known plasmid sequence from raw WGS data. In order to comprehensively analyze plasmids, complete plasmid DNA sequencing provides a powerful tool for obtaining the full plasmid sequence.

Complete plasmid sequencing

Complete plasmid sequencing utilizes the power of next-generation sequencing (NGS), PacBio sequencing or nanopore sequencing to obtain all sequences of any plasmid. It allows complete characterization of unknown plasmids and complete validation of known plasmids. This method does not require primers or rely on reference databases. The workflow for complete plasmid sequencing includes plasmid DNA preparation and quality control (QC), library construction and QC, sequencing, and bioinformatics analysis (including data QC, de novo assembly and further analysis). For NGS, it is difficult to achieve contiguous assemblies of plasmids as the read length, typically less than ∼400 to 500 bp, cannot cover the full-length of most mobile genetic elements. Thus, assemblies of plasmids from NGS data are often fragmented or incomplete. Third-generation sequencing, also known as long-read sequencing, includes PacBio and Nanopore sequencing, delivering rapid plasmid sequencing and allowing full assembly of plasmids to closure in many cases.

Full plasmid sequencing allows the identification of the genetic changes behind bacterial plasmid adaptation to new hosts or habitats as well as the co-evolution of bacterial genomes and plasmids during the introduction of new genes. The introduction of a large amount of DNA may have an impact on the host’s metabolism and fitness, which may be solved by different evolutionary strategies. Monitoring the sequence of plasmids can ensure your research progress and accelerate downstream applications, and is essential for accurate epidemiologic tracking of both routine surveillance and hospital outbreaks.

References:

1. Roosaare M, Puustusmaa M, Möls M, et al. PlasmidSeeker: identification of known plasmids from bacterial whole genome sequencing reads. PeerJ, 2018, 6: e4588.
2. Gutiérrez-Barranquero J A, Cazorla F M, de Vicente A, et al. Complete sequence and comparative genomic analysis of eight native Pseudomonas syringae plasmids belonging to the pPT23A family. BMC genomics, 2017, 18(1): 365.
3. Jackson R W, Vinatzer B, Arnold D L, et al. The influence of the accessory genome on bacterial pathogen evolution. Mobile genetic elements, 2011, 1(1): 55-65.
4. Bogas D, Nyberg L, Pacheco R, et al. Applications of optical DNA mapping in microbiology. BioTechniques, 2017, 62(6): 255-267.
5. Lemon J K, Khil P P, Frank K M, et al. Rapid nanopore sequencing of plasmids and resistance gene detection in clinical isolates. Journal of clinical microbiology, 2017, 55(12): 3530-3543.
6. Hu H, Manos J. Pulsed-field gel electrophoresis of Pseudomonas aeruginosa//Pulse Field Gel Electrophoresis. Humana Press, New York, NY, 2015: 157-170.