Complete Plasmid DNA Sequencing

CD Genomics provides complete plasmid DNA sequencing service, and our improved bioinformatics pipelines are available to perform de novo assembly with no reference required.

What Is Complete Plasmid DNA Sequencing

Plasmids are perceived as mobile genetic elements that exist extra-chromosomally and occasionally carry accessory genes that confer an advantage to their host in its ecological niche. They are thus thought to play an important evolutionary role in microbial communities by laterally introducing genes and traits into microbial genomes. Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness towards environmental change. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens.

Despite their importance, technical obstacles still limit the plasmid study. Plasmid DNA sequencing is rapidly becoming a standard approach to increase our understanding of the genetic diversity and evolutionary history of plasmids. Complete plasmid genome sequencing refers to the process of determining the entire genetic sequence of a plasmid. It not only helps to define the molecular events that took place during the evolution of these plasmids, but also give us a more complete overview of the enormous collection of accessory genes encoded on plasmids. And comparative plasmid sequence analysis has provided insights into the evolution of plasmids and their relatedness, their modular structure and the existence of hot spots for the insertion of accessory genes.

Our Full length Plasmid DNA Sequencing Methods

Plasmids exhibit significant heterogeneity in terms of size and complexity, often featuring high GC content, intricate structures, or large repetitive segments. These characteristics have historically rendered traditional Sanger sequencing methods cost-prohibitive, prone to high failure rates, and characterized by relatively slow processing.

Whole Plasmid Sequencing vs. Sanger Sequencing

Aspect Sanger Sequencing Whole Plasmid Sequencing (NGS)
Methodology Chain-termination method Utilizes Next-Generation Sequencing (NGS)or Long Read Sequencing technologies
Sequence Length Suitable for shorter fragments (up to 1,000 bp) Can sequence entire plasmids, regardless of size or complexity
Speed Sanger Sequencing can involve extended processing times and is particularly suited for small-scale projects. In contrast, Whole Plasmid Sequencing using NGS technologies provides a swifter turnaround, making it well-suited for high-throughput applications.
Cost Can be cost-prohibitive for larger plasmids due to multiple sequencing reactions Generally more cost-effective, especially for larger plasmids, as it involves fewer sequencing reactions
Accuracy Sanger Sequencing is renowned for its exceptional precision and minimal error rates. Whole Plasmid Sequencing maintains a high degree of accuracy and incorporates enhanced error correction methods.
Applicability Sanger Sequencing is appropriate for smaller plasmids but may encounter challenges when dealing with larger, intricate, or repetitive plasmid structures. Whole Plasmid Sequencing is highly versatile and can accommodate plasmids of various sizes and complexities, rendering it suitable for comprehensive plasmid analysis.

At CD Genomics, we carry out a thorough examination of plasmid genomes using cutting-edge next-generation high-throughput sequencing technology. Our method includes both library creation and sequencing, and it integrates state-of-the-art bioinformatics tools to provide a thorough analysis of sequencing depth, GC content, and the full assembly of plasmid sequences.

Moreover, we deploy a unique long-read assembly protocol in conjunction with state-of-the-art sequencing technology. This innovative combination affords an expedient, economical, and highly efficient avenue for the singular assembly of plasmids spanning various length spectrums. This novel approach not only facilitates an all-encompassing comprehension of plasmid content but also establishes it as the foremost scientific tool in the landscape of plasmid research.

Its utility transcends scientific boundaries, finding application in a multitude of interdisciplinary fields. These encompass the domains of synthetic biology, the nuanced regulation of gene expression, the precision of genome editing, the thorough exploration of bacterial antibiotic resistance mechanisms, and the meticulous scrutiny of various trait-associated phenomena. Furthermore, this method demonstrates its prowess in the realms of cellular therapy, the advancement of pharmaceuticals, and the enhancement of medical diagnostics, underscoring its invaluable contribution to the scientific community.

Key Features and Advantages of Complete Plasmid DNA Sequencing

  • High-Quality Data & Swift Turnaround For large plasmids or plasmids lacking known reference sequences, Sanger sequencing and NGS sequencing typically entail multiple sequencing attempts, whereas complete plasmid genome sequencing can often be accomplished within a short timeframe, sometimes requiring just a few working days.
  • Cost-Effective High-Throughput Sequencing The larger and more numerous the plasmids, the lower the cost. Sequencing large plasmids exceeding 60,000 base pairs can be achieved at less than one-tenth the cost of traditional methods, significantly less than Sanger sequencing.
  • Precise Assembly Compared to standard plasmids, alignment accuracy exceeds 99.9%.
  • High Success Rate For plasmids without reference sequences, both NGS and traditional Sanger sequencing suffer from high failure rates, especially in cases involving plasmids with high GC content or extensive repetitive structural elements. Complete plasmid genome sequencing does not require reference sequences and can achieve successful assembly in a single attempt.
  • High Automation Level Automated assembly analysis eliminates the need for manual peak profile interpretation, reducing the potential for errors.
  • User-Friendly Results Assembly files can be directly aligned, while variation files resemble peak profiles, allowing for straightforward data interpretation.

Complete Plasmid DNA Sequencing Workflow

Workflow Diagram of Complete Plasmid DNA Sequencing.

Service Specifications

Sample RequirementsComplete Plasmid DNA Sequencing:
  • Plasmid DNA≥ 1 µg;
  • Minimum Quantity: 500 ng;
  • Concentration ≥ 20 ng/µL
Plasmid DNA Long Read Sequencing:
  • Genomic DNA≥ 1 µg;
  • Minimum Quantity: 500 ng;
  • Concentration ≥ 10 ng/µL
Note: Sample amounts are listed for reference only. For detailed information, please contact us with your customized requests.
Sequencing Strategies
Data Analysis We provide multiple customized bioinformatics analyses:
  • Data quality control
  • High quality data acquisition
  • Genome de novo assembly
  • Detection of plasmid-specific sequences
  • …and more
Note: Recommended data outputs and analysis contents displayed are for reference only. For detailed information, please contact us with your customized requests.

Analysis Pipeline

The Data Analysis Pipeline of Complete Plasmid DNA Sequencing.

Deliverables

  • FASTQ files
  • Read-length and quality report
  • Assembly results
  • Plasmid circular map
  • Analysis of potential mutations
  • Plasmid annotation files

CD Genomics's plasmid sequencing service utilizes the power of Illumina Platform for plasmid verification, and has developed a low-cost high-throughput approach for complete larger plasmid sequencing using nannopore technology. We are pleased to use our extensive experience and advanced platform to offer the best service and the most qualified products to satisfy each demand from our customers.

The Complete Plasmid DNA Sequencing Results Display Figure.

1. Why is complete plasmid DNA sequencing necessary?

  • Validating sequence integrity: Plasmids may undergo genetic modifications during cloning or amplification, introducing potential errors or mutations.
  • Quality control for cloning and engineering: Sequencing plasmids ensures accurate verification of cloned inserts, absence of mutations, and experimental system integrity.
  • Optimizing experimental design: Accurate plasmid sequences enable effective experimental planning.

2. How to extract plasmid DNA from host cells?

Common extraction methods include:

  • Alkaline lysis: Cell lysis under alkaline conditions to release plasmid DNA.
  • Phenol-chloroform extraction: Utilizing phenol and chloroform for DNA extraction, eliminating proteins and impurities.
  • Column-based extraction: Commercial columns for plasmid DNA purification through centrifugation and elution.

3. How to ensure sequencing data accuracy?

Critical measures to ensure the precision of sequencing data encompass several key procedures:

  • Thorough sample extraction: Guaranteeing the purity and concentration of plasmid DNA.
  • Selecting a suitable sequencing platform: Tailoring the choice to experimental demands.
  • Implementing data processing and quality control: Employing bioinformatics tools to eliminate substandard reads and sequencing errors.
  • Validating through sequence alignment: Aligning the results with known sequences to substantiate accuracy.

4. What are the applications of complete plasmid DNA sequencing?

Complete plasmid DNA sequencing boasts a plethora of applications across various domains:

  • Gene cloning and expression studies: Facilitating the construction of effective cloning vectors.
  • Antibiotic resistance research: Unraveling bacterial resistance mechanisms.
  • Gene function studies: Delving into specific gene functionalities within cells.
  • Synthetic biology and biotechnology: Pioneering the design of synthetic biology tools and pathways for pharmaceutical production, environmental restoration, and bioenergy ventures.

If you would like to learn more, please refer to our article "Plasmid Detection and Complete Plasmid DNA Sequencing."

5. What factors may affect the results of plasmid DNA sequencing?

Influencing factors encompass:

  • Sample quality: DNA purity and concentration adequacy.
  • Sequencing technology: Appropriateness of chosen platforms and technologies.
  • Data processing: Effectiveness of quality control and processing procedures.
  • Sequence complexity: Impact of plasmid sequence complexity and repetitive elements on assembly.

Dynamics of Antimicrobial Resistance and Genomic Epidemiology of Multidrug-Resistant Salmonella enterica Serovar Indiana ST17 from 2006 to 2017 in China

Journal: Msystems

Impact factor: 7.324

Published: 21 July 2022

Background

Nontyphoidal Salmonella enterica (NTS) is a significant global foodborne pathogen, increasingly associated with antimicrobial resistance (AMR), especially multidrug-resistant (MDR) strains. This resistance complicates treatment and poses a public health challenge, leading the WHO to prioritize resistant S. enterica for new antimicrobial development. The genetic basis of resistance includes chromosomal mutations and plasmid-mediated genes, contributing to the widespread dominance of certain strains. Recent studies in China highlight the prevalence and resistance mechanisms of MDR S. Indiana, underscoring the urgent need for effective interventions to curb its spread.

Methods

Sample Preparation:
  • 251 S. Indiana isolates
  • Fecal samples
  • Clinical samples and food-related samples
  • Antimicrobial susceptibility testing
  • DNA extraction
Sequencing:
Data Analysis:
  • Analysis of blaCTX-M genomic locations
  • Phylogenetic analysis

Results

Among 138 blaCTX-M-positive isolates, 65.2% were classified by genome location. Notably, blaCTX-M-14 and blaCTX-M-55 were identified on chromosomes, while blaCTX-M-15 and blaCTX-M-65 were predominantly carried by plasmids. Human isolates exhibited a significantly higher prevalence of blaCTX-M positivity (63%) compared to food-related isolates (47%). Complete sequencing of five representative isolates unveiled a diverse landscape of genetic contexts harboring blaCTX-M genes, particularly on IncHI2 plasmids. Remarkably, plasmid pIndS104-CTX displayed striking similarity to a chromosomal locus in S. Indiana SI43, implying potential recombination events between plasmids and chromosomes. This study underscores the complexity of blaCTX-M dissemination pathways among pathogens and emphasizes the urgent need for further elucidation.

Fig 1. Circular comparison of blaCTX-M-positive IncHI2 plasmids identified in this study (pIndS104-CTX, ps12177-CTX, and ps11011-CTX) with other analogous IncHI2 plasmids cataloged in the NCBI nr database. (Du et al., 2022)Fig 1. Circular comparison between blaCTX-M-positive IncHI2 plasmids in this study (pIndS104-CTX, ps17177-CTX, and ps11011-CTX) and other similar IncHI2 plasmids in the NCBI nr database.

The study revealed that 56.2% of isolates were resistant to cefotaxime, with various CTX-M variants detected in S. Indiana, influencing their resistance phenotypes. Notably, blaCTX-M-65 was the most widespread, followed by blaCTX-M-14/55, contrasting with reports from Vietnam. This pattern aligns with previous findings in China, highlighting blaCTX-M-65 and blaCTX-M-14 as prevalent genotypes conferring cefotaxime resistance. Additionally, blaCTX-M-15 was exclusively found in human S. Indiana isolates, indicating distinct transmission routes. The blaCTX-M-55 variant, associated with enhanced cephalosporin-hydrolyzing activity, exhibited increased prevalence, particularly in lineage 6, suggesting heightened adaptability. IncHI2 plasmids were identified as typical carriers of blaCTX-M, mcr-1, and other resistance genes, facilitating transmission between Salmonella and other species. Moreover, the study identified mph(A)-positive isolates predominantly in lineages 5 and 6, coexisting with diverse ESBL genes, possibly mediated by IncX plasmids. Plasmids with various replicons (IncA/C, B/O, HI1, HI2, I1, N, F, and P) were associated with multidrug resistance in Salmonella, presenting challenges to public health. Furthermore, differences in antimicrobial susceptibility phenotypes and AMR gene profiles were observed among phylogenetic lineages and isolates from different sources, underscoring the complexity of transmission dynamics via the food chain.

Conclusion

This study offers a detailed insight into the rapid evolution of multidrug resistance (MDR) in S. Indiana over the past 15 years in China. Unique antimicrobial resistance mechanisms distinguish S. Indiana from other serovars, with diverse genetic processes contributing to resistance development, including chromosomal integrations, evolution of mobile resistance elements, and sporadic acquisition of resistance determinants. The presence of diverse host niches, including various animal reservoirs, underscores the importance of a One Health approach for efficient monitoring and control of resistance spread. Continuous surveillance targeting bacterial strains and mobile genetic elements is essential for effective control measures.

Reference:

  1. Du P, Liu X, Liu Y, et al. Dynamics of antimicrobial resistance and genomic epidemiology of multidrug-resistant Salmonella enterica serovar Indiana ST17 from 2006 to 2017 in China. Msystems, 2022, 7(4): e00253-22.
For Research Use Only. Not for use in diagnostic procedures.
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