How are samples sequenced and how accurate is the sequencing?

Samples undergo quality control to check concentration, purity, and total amount. Once samples meet library construction standards, a PCR-free library is created using a library preparation kit. Circular plasmids are linearized, barcoded, and combined to form the library. This library is then sequenced. Our proprietary assembly process efficiently aligns and assembles sequencing data into a high-quality, consistent sequence. The accuracy of sequencing is significantly enhanced, with a comparison to standard references achieving over 99.9% consistency.

Why is it necessary to provide both forward and reverse primer sequences and plasmid length information?

Although plasmids are typically derived from monoclonal samples, there may be contamination during extraction, potentially resulting in multiple assembly outcomes. To provide accurate results for the intended clone, specific primer sequences and plasmid length information are required to ensure correct matching and confirmation of the target sequence before delivering the results.

What if my sample concentration is below the required threshold?

Low concentration samples may result in inadequate representation during pooling, leading to insufficient sequencing depth or assembly failure. For samples below 5 ng/µL, we recommend resubmission. If you still wish to proceed, the sample will be processed with a risk-based approach and attempted only once. Regardless of the outcome, the full fee will apply.

Tel: 1-631-338-8059 Fax: 1-631-614-7828 Email: info@cd-genomics.com Inquiry

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Microbial Functional Gene Analysis
Microbial Functional Gene Analysis Service by NGS
Plasmid Identification - Full Length Plasmid Sequencing

Plasmid Identification - Full Length Plasmid Sequencing

Service Overview
Service Specifications
Demo
FAQs
Customer Case

Service Overview

Plasmid-mediated horizontal gene transfer is considered a major driving force for rapid bacterial adaptation and diversification. Identification of unknown or known plasmids was once difficult due to repeat regions. CD Genomics employs next generation sequencing (NGS) or long-read sequencing technology to accurately obtain all genomic knowledge of plasmid. Coupled with powerful bioinformatics algorithms and tools, we can help you identify antibiotic resistance genes (ARGs), mobile genetic elements and any other genes of interest.

Our Advantages:

Enhanced Efficiency: Significantly shortening experimental timelines, particularly for large plasmids and challenging plasmids without reference sequences, contributes to heightened overall efficiency.
Cost-Effectiveness: The cost of whole plasmid sequencing is substantially lower than Sanger sequencing, offering a cost-effective alternative with high value for money.
High Accuracy: Whole plasmid sequencing exhibits a matching accuracy of over 99.9% when aligned against standard quality plasmids, ensuring the reliability of experimental outcomes.
Increased Success Rate: For plasmids lacking reference sequences, the approach enables a one-time successful assembly, thereby elevating the overall success rate.
High Automation: Leveraging automated assembly analysis in whole plasmid sequencing reduces errors associated with manual peak chart interpretation, consequently enhancing sequencing accuracy.
User-Friendly Results: Assembly files are directly comparable, and variant files resemble peak charts, facilitating straightforward data assessment and thereby improving the efficiency of data processing.

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What Is Whole Plasmid Sequencing

Plasmids are independent DNA molecules found in bacteria, yeast, and filamentous fungi, separate from the chromosomal DNA in the nucleus. Situated in the cytoplasm, they replicate autonomously and consistently in offspring cells, expressing their genetic content. Plasmids are versatile tools in genetic engineering, existing in linear or circular forms. They can transfer between cells, promoting the swift spread of accessory genes among bacterial populations. Agencies like the European Food Safety Authority (EFSA) and the U.S. Food and Drug Administration (FDA) require comprehensive plasmid information in the food and feed sectors due to their role in antibiotic resistance gene transmission.

Whole-plasmid sequencing is a technique that utilizes both NGS and long-read sequencing technologies to obtain the complete sequence of an entire plasmid genome. By constructing a library of the plasmid DNA, the sequencing process generates a continuous sequence that spans the entire plasmid without fragmentation. This approach leverages advanced long-read sequencing technologies, such as those from Oxford Nanopore Technologies, to produce comprehensive data on the plasmid's genetic content in a single read. The resulting sequencing data is then analyzed bioinformatically to assess various parameters, including sequencing depth, GC content, and the assembly of the full plasmid sequence.

Differences Between Whole Plasmid Sequencing and Sanger Sequencing

	Sanger Sequencing	Whole Plasmid Sequencing
Data Quality	Peaks, double peaks; requires manual analysis and correction	Superior quality; no need for manual correction
Sequencing Length	1 kb	2.5 - 300 kb
Cost	Low	Lower
Sample Quantity	100 ng	1-100 ng
Accuracy	70-90%	≥97%
Sequencing Capability	Requires sequencing primers; limited to sequencing primer-targeted fragment sequences	No need for sequencing primers; capable of sequencing the entire plasmid length
Throughput	Low	High
Experimental Timeline	Several tens of days	A few days

Applications of Whole Plasmid Sequencing

Whole Plasmid Sequencing is applied to, but not limited to, the following areas:

Gene Synthesis Verification
Transgene Verification
Gene Editing Verification
Expression Regulation Verification
Vector Construction Verification
Synthetic Biology
Antibiotic Resistance Plasmid Identification

Service Specifications

Introduction to Our Whole Plasmid Sequencing Service

NGS and long-read sequencing technologies, such as Illumina HiSeq, PacBio, and Oxford Nanopore platforms, hold substantial promise for plasmid identification, diversity assessment, characterization, and evolutionary studies. Our commitment is to provide full-length plasmid sequencing services, enabling the detection and precise identification of plasmids. Long-read sequencing techniques allow for the comprehensive resolution of complex repetitive regions, thereby ensuring complete and accurate sequence data.

Through our services, you can uncover mobile genetic elements, screen for antibiotic resistance genes (ARGs), and annotate additional accessory genes. Accurate annotation of plasmid genomes is imperative to maximize the utility of the burgeoning dataset of bacterial strains. These detailed insights are crucial for determining food safety and quality, as well as for controlling antimicrobial resistance.

Whole Plasmid Sequencing Workflow

The Workflow of Whole Plasmid Sequencing.

Technical Parameters

Sequencing Platform	Library Type	Sequencing Depth
Illumina 150PE/250PE	400~500 bp	100X
PacBio SMRT	2 K	100X
Nanopore	-	-

Bioinformatics Analysis

Our bioinformatics analysis includes data quality control, genome assembly, functional annotation and comparative genomic analysis, which is flexible to your needs. Please feel free to contact us to discuss your project.

Bioinformatics Analysis	Details
Data QC	Data resource: Illumina (PE 75/250, 100X) or PacBio (4 Kb library, 100X)
Data QC	Read quality assessment, trimming and filtering
Sequence assembly	De novo assembly and outcome evaluation
Functional Annotation	NCBI GenBank, SwissProt, ARDB (Antibiotic Resistance Genes Database), COMBREX, Snapgene, PlasMapper, etc.

Note: The above content includes only a portion of the bioinformatics analysis. For more information or to customize the analysis, please contact us directly.

The Bioinformatics Analysis pipeline of Whole Plasmid Sequencing

Sample Requirement

Sequencing Platform	Sample Type	Recommended Quantity	Minimum Quantity	Minimum Concentration
Illumina	Plasmid DNA	≥ 1 µg	500 ng	10 ng/µL
Nanopore	Genomic DNA	≥ 1 µg	500 ng	10 ng/µL

1.8 < OD260/280 < 2.0, no degradation or contamination.

Note: If you wish to obtain more accurate and detailed information regarding sample requirements, please feel free to contact us directly.

Deliverables

Raw sequencing data
Assembled and annotated sequences
Quality-control dashboard
The customized bioinformatics report

Demo

Partial results of our Whole Plasmid Sequencing service are shown below:

The Whole Plasmid Sequencing Results Display.

FAQs

Whole Plasmid Sequencing FAQ

1. How are samples sequenced and how accurate is the sequencing?
- Samples undergo quality control to check concentration, purity, and total amount. Once samples meet library construction standards, a PCR-free library is created using a library preparation kit. Circular plasmids are linearized, barcoded, and combined to form the library. This library is then sequenced. Our proprietary assembly process efficiently aligns and assembles sequencing data into a high-quality, consistent sequence. The accuracy of sequencing is significantly enhanced, with a comparison to standard references achieving over 99.9% consistency.
2. Why is it necessary to provide both forward and reverse primer sequences and plasmid length information?
- Although plasmids are typically derived from monoclonal samples, there may be contamination during extraction, potentially resulting in multiple assembly outcomes. To provide accurate results for the intended clone, specific primer sequences and plasmid length information are required to ensure correct matching and confirmation of the target sequence before delivering the results.
3. What if my sample concentration is below the required threshold?
- Low concentration samples may result in inadequate representation during pooling, leading to insufficient sequencing depth or assembly failure. For samples below 5 ng/µL, we recommend resubmission. If you still wish to proceed, the sample will be processed with a risk-based approach and attempted only once. Regardless of the outcome, the full fee will apply.

Please feel free to reach out if you have any further inquiries or require additional information.

Customer Case

Host-specific plasmid evolution explains the variable spread of clinical antibiotic-resistance plasmids
Journal: Proceedings of the National Academy of Sciences
Impact factor: 9.412
Published: April 6, 2023

Find out more

Backgrounds

Plasmids are key genetic elements in bacteria, providing traits like antibiotic resistance and virulence factors. Their spread and stability are influenced by growth costs, transfer rates, and evolutionary changes. This study examines whether the variation in plasmid stability traits and their evolution during the absence of antibiotics affect the persistence of resistance plasmids. Using clinical E. coli strains and their associated plasmids, researchers tracked plasmid frequencies and stability traits, revealing that rapid evolution can alter plasmid dynamics, often overriding initial stability traits and demonstrating strain-specific evolutionary patterns.

Materials & Methods

Sample preparation:

Bacterial strains

Method:

WGS
Illumina MiSeq
Oxford Nanopore MinION

Data Analysis:

Quality control
Quality filtering
Read mapping

Results

Whole-genome sequencing revealed that genetic changes in ESBL plasmids and bacterial chromosomes are specific to the strain-plasmid combination. Evolved clones showed parallel mutations linked to increased plasmid transfer rates and interactions with mobile elements. Chromosomal changes, including deletions of pathogenicity islands, varied with the strain and plasmid, affecting bacterial growth.

Fig 1. Genetic alterations in chromosomes and ESBL plasmids following 15 days of serial passage without antibiotics. (Benz et al., 2023) Fig 1. Genetic changes in chromosomes and ESBL plasmids after 15 d of serial passage without antibiotics.

Evolved p1ESBL and p15ESBL plasmids in strain 15 showed increased transfer rates due to mutations in the plasmid leading region, specifically in the ssi3 operon. These mutations, including stop codon SNPs and frame shifts, enhanced transfer rates in strain 15 but not in other strains, indicating a strain-specific effect. The findings suggest that mutations in this region may be a common adaptation mechanism for improving plasmid transfer in clinical settings.

Fig 2. Host-specific increases in transfer rate due to genetic changes in the plasmid leading region. (Benz et al., 2023) Fig 2. Genetic changes in the plasmid leading region increase transfer rate in a host-specific way.

Conclusions

The authors found that plasmid persistence is driven more by rapid, strain-specific evolutionary changes than by initial stability traits. Mutations in the plasmid leading region, particularly in the ssi3 operon, affect transfer rates differently across strains. Their study highlights the importance of considering rapid evolution in managing successful bacterium-plasmid combinations and suggests further research to refine predictions in clinical settings.

References

Thomas C M, Thomson N R, Cerdeno-Tarraga A M, et al. Annotation of plasmid genes. Plasmid, 2017, 91: 61-67.
Benz F, Hall AR. Host-specific plasmid evolution explains the variable spread of clinical antibiotic-resistance plasmids. Proceedings of the National Academy of Sciences. 2023, 120(15):e2212147120.

* For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.

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Tel: 1-631-338-8059
Fax: 1-631-614-7828
Email: info@cd-genomics.com

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