NGS Sequencing Methods: A Comprehensive Beginner's Guide
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Highlights
Learn the basics of Next-Generation Sequencing (NGS) and discover how MGI’s DNBSEQ™ technology offers better, faster, stronger, and more affordable sequencing solutions than ever before.
What Is Next-Generation Sequencing?
Next-Generation Sequencing (NGS) is a transformative technology that has revolutionized genomics by enabling scientists to sequence DNA and RNA rapidly and accurately with high throughput. The entry of new players in the market, offering alternative technologies and more choices for users, has significantly reduced costs per gigabase in recent years, making this powerful technology accessible to more people.
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Key Advantages of Next-Generation Sequencing (NGS) Compared to Traditional Methods
Compared to microarrays or PCR, NGS allows researchers and clinicians to explore genetic complexity with greater sensitivity, flexibility, and higher throughput across a range of applications. NGS has opened new avenues for discoveries in life sciences, providing valuable insights for understanding biological processes, identifying causes of diseases, detecting microorganisms, and characterizing phenotypes.
Methods of NGS
Whole Genome Sequencing (WGS): Sequencing the entire genome of an organism, offering a comprehensive view of both coding and non-coding regions.
Whole Exome Sequencing (WES): Focusing on sequencing all exons (the exome), which are the genome's protein-coding regions.
Targeted Sequencing (Gene Panel Sequencing): Sequencing specific genes or regions of interest.
RNA Sequencing (RNA-Seq): Sequencing the entire transcriptome (all RNA in an organism), providing insights into gene expression patterns. Advanced technology now allows RNA-Seq at the single-cell level (scRNA-seq) and spatial level (spatial transcriptomics), adding new layers of information related to gene expression.
Epigenetic Sequencing: Examining modifications, such as DNA methylation, that affect gene activity without altering the DNA sequence.
Proteomics: NGS-based proteomics enables precise protein quantification.
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Vocabulary and Definitions
Coverage/Depth of Sequencing: The number of times a base is read in an NGS experiment. Higher coverage increases accuracy by ensuring each part of the genome is sequenced multiple times.
Flow Cell: A small device in sequencing instruments where DNA/RNA samples are placed and sequenced. Millions of DNA fragments are sequenced simultaneously in a flow cell.
Library Preparation: The process of preparing DNA or RNA samples for sequencing, including fragmentation, adapter ligation, and amplification.
Read: A DNA or RNA sequence generated by a sequencing machine. Reads are short fragments that are assembled to reconstruct the full sequence.
Read Length: The number of base pairs in a single read generated by a sequencing machine. Read length can vary depending on the sequencing platform, from short (50–300 bp) to long (up to several thousand bp). Longer reads provide more context for assembling complex genome regions, while shorter reads are more cost-effective and suitable for high-throughput applications.
Basic NGS Workflow
Embarking on NGS can seem daunting, but with the right approach, you can integrate this powerful technology into your lab.
1. DNA and RNA Extraction
Isolate high-quality DNA or RNA for optimal results, ensuring you have enough material for the library preparation step. The required quantity depends on the NGS method and library preparation kit used.
2. Library Preparation
Fragment the nucleic acids into sizes optimized for your library preparation kit and NGS method. These fragments are then amplified, typically by PCR or RCR (Rolling Circle Replication). Note that PCR amplification may introduce more errors than RCR. After generating your library, ensure it passes quality control (QC) for quality and quantity.
3. Sequencing
Once your library passes QC, load it onto a flow cell, insert the reagent cartridge, and start the sequencing run. Depending on the sequencer, you can run 2 to 4 flow cells independently.
4. Data Analysis
Analyzing NGS data requires bioinformatics expertise or specialized software that can interpret sequencing data without requiring coding skills.
Visual Workflow:
1
DNA, RNA extraction
2
Library preparation
3
Sequencing
3
Data analysis
What are the main differences between PCR and RCR
What MGI Can Offer You
Advanced Sequencing Platforms: High accuracy, adapted to your throughput needs.
Comprehensive Support: Expert guidance from sample preparation to data analysis.
Cost-Effective Solutions: Competitive pricing without compromising quality.
Innovative Technology: DNBSEQ™ technology lowers error rates, providing highly accurate data.
Compatibility with Library Preparation Kits: Compatible with providers such as Twist, IDT, NEB, and more.
Global Presence: Serving researchers worldwide with cutting-edge technology.
Complete NGS Workflow: From sample extraction to data analysis.
Explore our complete NGS workflow
By understanding the basics of NGS and considering these key factors, you can make informed decisions that will advance your research and clinical applications. MGI is here to support you every step of the way.
Considerations Before Investing in an NGS Platform
Applications: Depending on your applications and NGS methods, you will use specific read lengths. Ensure your sequencer can accommodate these requirements, and consider flexibility if using multiple NGS methods.
Project Scale: Choose a platform that matches your sample throughput needs.
Budget: Factor in not only the cost of the instrument but also expenses for reagents and consumables.
Resources:
Bioinformatics Team: Do you have a bioinformatics team for data analysis? If not, software solutions can help analyze sequencing data without coding expertise.
Data Storage: Sequencing generates a large amount of data. Ensure you have the infrastructure to store it. Storing your data in the cloud might also be a possibility to explore.
Automation: Automating extraction and library preparation can save time and costs, freeing up your team for other lab tasks.
Technical Support: Access to a local, accessible technical and scientific support team is essential. Ensure you have access to this kind of support.
DNBSEQ-G99
DNBSEQ-G99 is a high-speed NGS benchtop sequencer ideal for targeted gene and small genome sequencing. It offers flexible throughput (8-96 Gb per run), completing PE150 sequencing in just 12 hours with rapid preliminary reports.
