Sanger sequencing and NGS (next-generation sequencing) are two DNA analysis methods that allow for the precise identification of a sample's nucleotide sequence. Sanger sequencing, the historical method, is renowned for its high reliability on short sequences, while NGS enables massive, parallel sequencing of thousands of fragments, ideal for large-scale studies. In an industrial context where traceability, authentication, and compliance have become crucial, DNA sequencing is an essential tool. DNA levels ensures the appropriate quality and concentration for optimal sequencing. Understanding the differences, applications, and complementarity of Sanger and NGS is essential for making the right analytical choices in sectors as diverse as food and beverage, animal health, cosmetics, and the environment.
Table of Contents
Introduction: Two complementary approaches to DNA sequencing
This article aims to clarify the differences between Sanger sequencing and NGS sequencing, explaining their scientific principles, advantages and disadvantages, and concrete laboratory applications. It is intended for R&D, quality, and regulatory professionals who wish to better understand these technologies in order to make informed choices for their analytical projects.
We will also see how specialized laboratories like those in the YesWeLab network can support you in choosing the method, carrying out the analyses, and interpreting the results.
Scientific and industrial context
Since the publication of the human genome in the early 2000s, sequencing technologies have undergone rapid development. Today, genome, transcriptome, and microbiota analysis are integral to research and quality control strategies in many industrial sectors. Sequencing allows for the precise determination of a sample's genetic composition, the detection of mutations, verification of an ingredient's origin, and monitoring of the biological activity of a medium.
Faced with these growing needs, two main sequencing methods coexist. On the one hand, Sanger sequencing, developed in 1977, remains a reference method for short sequences and projects requiring maximum precision. On the other hand, next-generation sequencing (NGS), which emerged in the mid-2000s, allows for the simultaneous processing of millions of DNA fragments for complex, high-throughput analyses.
Challenges for manufacturers
In the food industry, sequencing techniques are used to authenticate the animal or plant species used in processed products, detect fraud (such as species substitution in fish or spices), and analyze microbial strains present in fermented foods. In animal health, they allow researchers to track the evolution of pathogens and study the gut microbiota. In the pharmaceutical and cosmetic sectors, sequencing can be used to identify medicinal plants or verify the conformity of bacterial strains.
For all these applications, choosing the right sequencing method is crucial. A poor choice can lead to incomplete results, unnecessary additional costs, or delays in product development.
Sanger sequencing: the historical reference method
Scientific principle of Sanger sequencing
Developed in 1977 by Frederick Sanger, Sanger sequencing relies on a method called chain termination. This technique uses dideoxynucleotides (ddNTPs), analogs of natural nucleotides (dNTPs) that block DNA elongation when incorporated into a strand. During the polymerization reaction, a mixture of normal dNTPs and ddNTPs labeled with fluorophores is used. Each type of ddNTP (A, T, G, C) emits a specific fluorescence.
The DNA thus synthesized, fragmented to different lengths, is then separated by capillary electrophoresis. By analyzing the fluorescent signals, the nucleotide sequence of a DNA fragment, generally between 100 and 1200 base pairs (bp), can be reconstructed.
This process is highly reproducible and accurate, which explains why it is still used today as a reference method, particularly for specific sequences or targeted checks.
Laboratory applications
Despite the emergence of NGS, Sanger sequencing remains essential in many situations. It is used in various industrial sectors and applied research, particularly for:
- Molecular barcoding : this method allows the identification of a species from a single sample by targeting a ubiquitous gene (such as COI for animals, 16S for bacteria, or rbcL for plants). Sanger sequencing is ideal for simple, isolated matrices.
- Mutation confirmation : after detection of a mutation by qPCR or NGS, the Sanger method is used to confirm the exact presence of the variation, particularly in TILLING projects on plants or in the context of precision veterinary medicine.
- Genetic construct verification : this allows for the validation of the sequence of a plasmid, vector, or modified gene. This includes verification in genetic engineering projects or recombinant protein production.
- Primer walking : in the case of long DNA fragments (> 1500 bp), the method consists of successively sequencing several overlapping regions using different primers to reconstruct the entire sequence.
- Raw material authentication : it can be used to verify that a plant extract, meat or fish corresponds to the expected species, in products based on a single species.
At YesWeLab, this technique is regularly used in partner laboratories for customized services in the agri-food, cosmetics and pharmaceutical sectors.
Advantages and limitations
The main advantage of Sanger sequencing lies in its accuracy , which exceeds 99.99%. This makes it a reliable method for targeted analyses requiring a high degree of confidence in the results. It is also simple to implement, with results available in 24 to 48 hours, and reasonably priced for small volumes.
Benefits :
- High fidelity reading
- Short deadlines
- Controlled costs for a small number of samples
- Proven method, compatible with all simple matrices
Boundaries :
- Low analysis volume (one fragment at a time)
- Unsuitable for complex samples or mixtures of species
- Low yield compared to NGS methods
- Requires a specific target sequence or primer known in advance
In summary, Sanger sequencing is the ideal solution for projects requiring high precision on a small number of genetic targets. For single-species analysis, mutation validation, or cloning verification, it remains a reliable and cost-effective option.
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NGS sequencing: high-throughput technology for complex matrices
How NGS sequencing works
Next-generation sequencing, or NGS , is based on a fundamentally different principle than Sanger sequencing. It allows for the simultaneous reading of millions of DNA or RNA fragments, thanks to a massively parallel sequencing . This ability to produce a large volume of data in a single operation makes it an essential technology for large-scale projects or for the analysis of complex matrices.
The procedure begins with DNA fragmentation, followed by library preparation : the fragments are ligated to adapters, then amplified and indexed. Depending on the platform used (Illumina, Ion Torrent, PacBio, Nanopore, etc.), the reading principle varies (by synthesis, ligation, ionic conductance, or electrical current variation), but all technologies aim to identify the exact order of nucleotides within each fragment.
The generated data are then converted into digital files (usually in FastQ ) that contain the sequences and the read quality of each base. These files must be analyzed using bioinformatics tools to perform alignments, assembly, variant searches, or taxonomic classifications.
Applications in industrial sectors
Next-generation sequencing (NGS) is particularly well-suited to projects with significant genetic or biological diversity, as well as situations requiring the simultaneous exploration of a large number of genes or organisms. Its applications are now widespread in industry.
Agri-food :
- Identification of species in processed foods, particularly to detect fraud or verify labeling (e.g., presence of horse meat in prepared dishes or undeclared species in fish mixtures).
- Analysis of the floral composition of honey by metabarcoding to determine its origin.
- Characterization of the microbiota in fermented products (yeasts, lactic acid bacteria…).
Environment :
- Assessment of microbial biodiversity in soils, biofilms, and aquatic environments.
- Ecological monitoring of watercourses by detection of diatoms, fungi, microinvertebrates.
- Measuring anthropogenic impacts on natural ecosystems by monitoring the metatranscriptome.
Pharmaceuticals and animal health :
- Traceability of medicinal plants used in food supplements (e.g., Aloe vera, Ginger, Neem).
- Study of the animal gut microbiota (dog, horse, poultry) to understand its influence on health and productivity.
- Detection of mutations or resistances in bacterial or viral genomes.
Cosmetic :
- Quality control of raw materials of plant or fermented origin.
- Study of the skin microbiome to develop products adapted to different skin types.
Concrete examples :
- A laboratory wishing to identify all microorganisms present in an agricultural soil can use NGS sequencing on the 16S region (bacteria) or ITS (fungi).
- An industrialist wishing to authenticate several species of spices in a mixture (e.g. saffron, turmeric, black pepper) would benefit from using NGS analysis by metabarcoding, which is more efficient than the Sanger method in this context.
Strengths and weaknesses
Next-generation sequencing (NGS) offers undeniable advantages when it comes to conducting in-depth genetic analyses or processing complex samples. Its ability to generate millions of reads in a single experiment paves the way for exhaustive exploration of a genome, transcriptome, or microbial ecosystem.
Benefits :
- Simultaneous analysis of thousands to millions of fragments
- Blind species identification , without prior hypothesis
- Cost decreases as the sample volume increases
- Targeted sequencing , WGS (Whole Genome Sequencing), or RNA-seq can be performed.
Constraints :
- Larger investment for small projects
- Longer processing time (bioinformatics required)
- Results are sometimes limited to the higher taxonomic rank if the DNA is degraded
- Need technical support for data interpretation
NGS sequencing is therefore a powerful technology, but one that requires strategic thinking about volumes, analytical objectives, and data processing. This is why many companies rely on platforms like YesWeLab to define their specifications, select the right technology, and analyze the results.
Which method should you choose based on your analysis objectives?
Typical use cases for Sanger sequencing
Sanger sequencing remains the preferred method when analyzing a targeted, well-defined sequence in a single or relatively simple sample. It is particularly relevant for confirmatory or validation analyses, especially when used in conjunction with other techniques such as qPCR or NGS.
Here are some concrete examples of the use of Sanger sequencing in an industrial context:
- Verification of point mutations : after detection of a mutation by NGS in a gene of interest (for example a resistance mutation in a bacterial gene), Sanger sequencing makes it possible to confirm or refute the variation, base by base.
- Validation of cloning and genetic constructs : in biotechnology projects, Sanger sequencing is used to ensure the integrity of vectors (plasmids, inserts), verify primers or identify sequence errors.
- Targeted species identification : to confirm the species of a plant (basil, mint, saffron) or animal (salmon, tuna) ingredient, provided the sample is not a mixture. This method is frequently used in quality control of raw materials in the food and cosmetics industries.
- Single-species sample analysis : if only one species is expected in the sample, such as a bacterial isolate or a dried leaf, Sanger is fast and economical.
In all these cases, the Sanger method is simple to implement, fast (24 to 48 hours) , and economical for a small number of samples.
Typical use cases for NGS sequencing
Next-generation sequencing (NGS) is the preferred method when dealing with a complex sample containing multiple species, or when exploring large genomic or transcriptomic regions. It excels in the comprehensive analysis of an ecosystem or a complete genetic profile.
Here are the main application cases:
- Authentication of complex mixtures : verification of the composition of a spice blend, a multi-component food supplement, or a prepared dish containing several species of fish or meat.
- Microbiota study : whether it is soil, water, animal intestine or skin, NGS makes it possible to identify all the microbial species present and to understand their interactions.
- Transcriptomics and metatranscriptomics : analysis of gene expression in a tissue or ecosystem. For example, in an animal intestine, NGS makes it possible to identify not only the species present, but also the genes they express.
- De novo sequencing or WGS : to sequence a complete genome that is still unknown (plant, bacterium, fungus), or to search for mutations in a known genome.
NGS is indispensable when the complexity, diversity, or depth of analysis exceeds the capabilities of Sanger.
Comparative table Sanger vs NGS
To help with decision-making, here is a summary table of the key differences between the two methods.
| Criteria | Sanger Sequencing | NGS Sequencing |
|---|---|---|
| Sample type | Single species, targeted sequence | Complex mixtures, multiple sequences |
| Data volume | Weak (one fragment at a time) | High (millions of simultaneous fragments) |
| Cost per sample | Low for small volumes | Profitable starting from large volumes |
| Delivery time | 24 to 48 hours | 3 to 7 days depending on bioinformatics required |
| Analysis complexity | Weak | High (bioinformatics analysis required) |
| Reading accuracy | Very high | Raised with sufficient cover |
| Blind identification | No | Yes (thanks to metabarcoding or WGS) |
| Common use | Confirmation, verification, barcoding | Metagenomics, transcriptomics, screening |
This table highlights the strategic complementarity between the two approaches. Ideally, in many projects, NGS should be used for exploration, followed by Sanger to confirm. This is particularly true in research projects, new product development, or enhanced quality control.
Scientific Focus: Laboratory Analyses and Genetic Sequencing
Laboratory techniques associated with sequencing
DNA sequencing, whether performed using the Sanger method or NGS, relies on a set of rigorous laboratory techniques, the reliability of which is essential to guarantee the quality of the results. These techniques may vary depending on the type of sample, the complexity of the matrix, and the method chosen, but generally follow a standardized workflow.
DNA extraction and purification
This is the essential first step. The goal is to isolate the DNA from the matrix being studied: plant, food, animal tissue, microorganism, water, soil, etc. This step must guarantee the purity and integrity of the DNA, as degradation or contamination can compromise the results. Depending on the matrix, the methods vary (mechanical grinding, enzymatic digestion, filtration, centrifugation) and can be automated.
PCR amplification
Polymerase chain reaction (PCR) allows for the targeting of a specific region of DNA and its amplification in large quantities. Specific primers are used for this purpose. In Sanger sequencing, this step produces the fragment to be analyzed. In next-generation sequencing (NGS), it is also used for barcoding or amplicon amplification. The choice of primers (universal, genus-specific, or species-specific) is a critical step for analytical reliability.
NGS Library Preparation
In the case of NGS sequencing, a DNA library . This involves fragmenting the DNA (if it is not already fragmented), ligating it to adapters, adding indexes (barcodes) to distinguish the samples, and checking the quality of the prepared fragments. This step is automated on some platforms (such as Ion Chef at CARSO) and uses precise enzymatic techniques.
Sequencing and bioinformatics analysis
The sequencing process itself depends on the technology used: capillary electrophoresis (Sanger), synthetic fluorescence (Illumina), ionic conductivity (Ion Torrent), or current reading (Nanopore). The data obtained must be analyzed using specialized bioinformatics tools. This includes read quality assessment, alignment with databases (BOLD, NCBI), mutation detection, and whole-genome reconstruction.
Standards and regulatory compliance
Analytical laboratories must operate according to very strict quality standards. In France, as in Europe, the ISO 17025 standards apply to testing and calibration laboratories. They guarantee the technical competence of staff, the traceability of procedures, the validation of methods, and the reliability of results.
COFRAC accreditation is an official recognition of a laboratory's compliance with these standards. It is an essential criterion of trust for industrial clients, particularly in sectors subject to stringent regulations such as food processing, animal health, and cosmetics.
In the context of sequencing, these standards govern all the steps:
- sample management,
- equipment maintenance (sequencers, thermocyclers),
- internal quality controls,
- and the issuance of reports that comply with regulatory expectations (information on the method, detection thresholds, quality of results, etc.).
YesWeLab works exclusively with partner laboratories that comply with ISO 17025 standards and are COFRAC accredited for the relevant services. This guarantees maximum reliability and regulatory validity for the results provided.
Examples of laboratory analyses related to sequencing
As with other analytical molecules such as malic acid, specific techniques are used in the laboratory to study the genetic components of a product. Here is a comparison between classical methods and those integrated into a sequencing workflow.
- High-performance liquid chromatography (HPLC) : used to analyze compounds such as malic acid, it is the equivalent, in terms of accuracy, of the Sanger method for a single sequence.
- Spectrophotometry : allows estimation of DNA concentration before or after extraction.
- Migration tests : in the context of materials in contact with food (e.g. packaging containing plant DNA), sequencing can confirm the nature of the raw material.
- Rheological tests : although not directly related to sequencing, they can be combined with genetic tests to validate the composition of a product (origin of the ingredient + physical behavior of the product).
Link between genetic analysis and regulation
In several sectors, genetic analysis is now a key tool for demonstrating regulatory compliance. In the food industry, it allows for verification of ingredient traceability according to the INCO regulation . In packaging, sequencing can confirm the biological nature of a material in accordance with EC Regulation No. 1935/2004 . In cosmetics, it validates the composition of natural raw materials, which is essential for brands claiming plant-based or organic formulas.
Thus, genetic analyses are increasingly being integrated into comprehensive quality management and are no longer reserved for research or high-level scientific testing. Thanks to platforms like YesWeLab, these methods are becoming accessible to manufacturers who want to control their supply chain, ensure product safety, and innovate.
Case studies: when Sanger or NGS sequencing is a game changer
Identifying a medicinal plant in a food supplement: the case of Sanger sequencing
A dietary supplement manufacturer wants to verify the authenticity of a plant extract marketed as containing exclusively Panax ginseng . The aim is to ensure labeling compliance and prevent any risk of fraud or unintentional substitution.
In this specific case, Sanger sequencing is the ideal method. The analysis begins with DNA extraction from the plant material. PCR amplification is performed targeting a specific gene such as rbcL or matK , commonly used for plant barcoding. The amplified fragment is then sequenced using the Sanger method and compared to international databases (NCBI, BOLD).
Result: The analysis confirms a 99.8% match with Panax ginseng . This result validates both the product's traceability and its conformity to the manufacturer's claims. The use of the Sanger test provided a rapid, precise, and legally binding result for quality control purposes.
Detecting multiple species in a cooked dish: the value of NGS sequencing
A prepared food retailer wants to ensure that the animal species used in a seafood gratin correspond to those listed on the label (mussels, shrimp, squid). It also suspects possible cross-contamination with undeclared species, such as crab or pollock.
In this type of complex matrix, Sanger sequencing is unsuitable: it can only detect one or two dominant species. Next-generation sequencing (NGS), on the other hand, allows for comprehensive identification. After DNA extraction, a PCR targeting a universal gene (e.g., COI for marine animals) is performed. The amplicons are then sequenced by NGS (Illumina or Ion Torrent platform), and the resulting sequences are analyzed to identify all the species present.
The analysis revealed the presence of the expected species, but also traces of Gadus morhua (cod) and Cancer pagurus (crab). These results allow the manufacturer to correct its recipe, adapt its cleaning procedures, and guarantee greater transparency for consumers.
Detecting variants in a plant culture: NGS and Sanger validation
A plant breeding applied research laboratory aims to identify point mutations in a gene of interest involved in water stress resistance in a wheat variety. The objective is to screen several hundred plants to select the mutant individuals.
The chosen strategy involves using NGS (exome or targeted region) sequencing to identify potential variants on a large scale, then validating these mutations by Sanger sequencing on the most promising plants. This combined approach leverages the power of NGS for mass detection while relying on the precision of Sanger sequencing for confirmation.
As a result, the laboratory identified several promising mutant alleles. Sanger sequencing confirmed their presence in selected individuals, which were then chosen for phenotyping tests. This combined approach optimizes the cost, accuracy, and efficiency of the breeding program.
Studying the gut microbiota of a racehorse: metabarcoding by NGS
A veterinary clinic specializing in equine performance wants to assess the composition of the gut microbiota of a competition horse suffering from recurrent digestive issues. The goal is to identify any bacterial imbalances that could affect the animal's health and performance.
Next-generation sequencing (NGS) is the essential method here. DNA is extracted from fecal samples, followed by PCR amplification targeting the V3-V4 region of the 16S gene (a universal bacterial marker). Sequencing is performed using Illumina MiSeq, and the data are then analyzed to establish a taxonomic profile of the bacterial community.
The analysis revealed dysbiosis, with an overrepresentation of Firmicutes and an underrepresentation of Bacteroidetes. This information led to a recommendation to adjust the diet and use targeted probiotics, with clinical improvement observed after a few weeks.
Quality control of a plasmid in genetic engineering: Sanger or long-read?
A biotechnology laboratory is developing a new cell line expressing a therapeutic protein. To validate the sequence of the inserted plasmid, two approaches are possible: Sanger sequencing (if the sequence is short and known) or long-read sequencing (if the plasmid is complex or contains repeated structures).
In this case, the company chose to use long-read sequencing with Nanopore , which allows for obtaining the complete plasmid sequence in a single read, including the backbone, promoters, and coding regions. This choice ensures complete validation of the genetic construct before industrial production.
These concrete examples demonstrate that the choice between Sanger, NGS, or long-read sampling depends directly on the complexity of the sample , the required level of precision , and the volume of information needed . Thanks to its network of partner laboratories, YesWeLab is able to adapt the method to each specific field challenge, guaranteeing quality, compliance, and efficiency.
Genetic testing services with YesWeLab
Multi-sector expertise at the service of your genetic analyses
YesWeLab offers comprehensive support for Sanger and NGS sequencing projects, regardless of the complexity of your samples or your industry. Through a network of over 200 accredited partner laboratories across France and Europe, the platform provides industry professionals with access to leading expertise in molecular biology, genomics, and bioinformatics.
The services are tailored to the specific needs of different sectors:
- Agri-food : species identification, verification of ingredient authenticity, microbiota analysis in fermented products.
- Nutraceuticals : verification of the composition of plant extracts, validation of origin claims.
- Animal health : intestinal microbiome analysis, pathogen detection, line genotyping.
- Cosmetics : authentication of natural raw materials, control of fermentation strains.
- Environment : monitoring of biodiversity in soils and aquatic environments, metabarcoding of complex samples.
- Pharmaceuticals and biotechnology : quality control of plasmid constructs, detection of genetic variants, transcriptomics.
YesWeLab acts as a single interface , simplifying the management of analytical projects from A to Z.
A digital platform to centralize your requests and results
One of YesWeLab's major strengths is its secure online platform , designed to centralize all stages of the analytical process:
- Search for analyses from a catalogue of over 10,000 services ,
- Direct ordering and traceability of sample shipments,
- Real-time monitoring of the analysis stages,
- Secure reception of results (raw sequences, chromatograms, analysis reports, bioinformatics files).
This solution is particularly appreciated by quality, R&D or regulatory affairs managers, who can thus efficiently manage several projects at once, while ensuring documentary compliance and complete data traceability .
Tailor-made services adapted to each sequencing method
YesWeLab offers Sanger and NGS sequencing services with different levels of intervention, depending on your needs in terms of accuracy, budget, and volumes.
For Sanger sequencing :
- Confirmatory analyses (mutations, plasmids, barcoding)
- Services with or without sample purification
- Single or double-direction reading, with manual or automatic corrections
- Results provided in .seq , .ab1 , or consensus sequence
For NGS sequencing :
- Amplicon sequencing (barcoding, metabarcoding)
- RNA-Seq, metatranscriptomics
- De novo sequencing or WGS
- Bioinformatics analyses: alignment, annotation, SNP detection, taxonomic profiles
Each service is customizable. YesWeLab can intervene from the design of your project interpretable and usable results .
Guarantees of quality, compliance and responsiveness
ISO 17025 certified or accredited laboratories and guarantees compliance with European (INCO, 1935/2004, REACH, etc.) and international (FDA, GMP) standards. Turnaround times are optimized: expect 24 to 48 hours for Sanger sequencing and 3 to 7 business days for NGS including bioinformatics analysis.
In the case of a complex request or a multidisciplinary project, a dedicated technical expert will support you throughout the process, from the validation of the specifications to the interpretation of the results.
Thanks to this approach, YesWeLab becomes a strategic partner for manufacturers concerned with making their analyses more reliable, accelerating their developments, and ensuring the quality of their products.
