Key differences between next-generation sequencing and Sanger sequencing

Understanding when NGS can be a more effective option

In principle, the concepts behind Sanger vs. next-generation sequencing (NGS) technologies are similar. In both NGS and Sanger sequencing (also known as dideoxy or capillary electrophoresis sequencing), DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand. Each incorporated nucleotide is identified by its fluorescent tag.

The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This process translates into sequencing hundreds to thousands of genes at one time. NGS also offers greater discovery power to detect novel or rare variants with deep sequencing.

Differences Between NGS and Sanger Sequencing

Advantages of NGS include:

  • Higher sensitivity to detect low-frequency variants1,2
  • Faster turnaround time for high sample volumes3
  • Comprehensive genomic coverage
  • Lower limit of detection4,5
  • Higher capacity with sample multiplexing
  • Ability to sequence hundreds to thousands of genes or gene regions simultaneously
Choosing NGS vs. Sanger Sequencing

Explore the benefits and limitations of each method to understand which one best suits your needs.

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“With Sanger sequencing, we saw a limited DNA snapshot… NGS and its massively parallel sequencing enable us to look at tens to hundreds of thousands of reads per sample.”

Michael Bunce, PhD
Professor, Head of TrEnD laboratory, Curtin University
  Sanger Sequencing Targeted NGS
Benefits
  • Fast, cost-effective sequencing for low numbers of targets (1–20 targets)
  • Familiar workflow
  • Higher sequencing depth enables higher sensitivity (down to 1%)
  • Higher discovery power*
  • Higher mutation resolution
  • More data produced with the same amount of input DNA
  • Higher sample throughput
Challenges
  • Low sensitivity (limit of detection
    ~15–20%)
  • Low discovery power
  • Not as cost-effective for high numbers of targets (> 20 targets)
  • Low scalability due to increasing sample input requirements
  • Less cost-effective for sequencing low numbers of targets (1–20 targets)
  • Time-consuming for sequencing low numbers of targets (1–20 targets)

* Discovery power is the ability to identify novel variants.
Mutation resolution is the size of the mutation identified. NGS can identify large chromosomal rearrangements down to single nucleotide variants.
10 ng DNA will produce ~1 kb with Sanger sequencing or ~300 kb with targeted resequencing (250 bp amplicon length × 1536 amplicons with an AmpliSeq for Illumina workflow)

Options for Sanger vs. Next-Generation Sequencing
 
Efficient Variant Discovery with Targeted Gene Panels
Efficient Variant Discovery with Targeted Gene Panels

NGS enabled Franco Taroni, MD to identify variants in a fraction of the time and at a significantly lower cost than Sanger sequencing.

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NGS Revolutionizes Reproductive Genomics
NGS Revolutionizes Reproductive Genomics

Viafet uses the VeriSeq PGS Solution, enabling IVF clinics to provide fast, accurate, and efficient PGS services.

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Environmental DNA Offers a Powerful Look at Biodiversity
Environmental DNA Offers a Powerful Look at Biodiversity

Sanger sequencing offered a "limited DNA snapshot." Now, Michael Bunce, PhD uses NGS to look at hundreds of thousands of reads per sample.

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Sanger sequencing can be a good choice when interrogating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer). Otherwise, targeted NGS is more likely to suit your needs. NGS allows you to screen more samples cost-effectively and detect multiple variants across targeted areas of the genome—an approach that would be costly and time-consuming using Sanger sequencing.

Watch this animation to see how the easy and accessible Illumina NGS technology can complement your Sanger sequencing work.

Cost-Effectiveness of Targeted NGS vs. Sanger Sequencing
Beginner's Guide to Next-Generation Sequencing
Beginner's Guide to Next-Generation Sequencing

In this introduction, we'll guide you through the steps in the NGS workflow and planning your first experiment.

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Targeted Resequencing
Targeted Resequencing

This method involves isolating and sequencing a subset of genes or a genomic region of interest, which can conserve lab resources.

Whole-Genome Sequencing
Advantages of Whole-Genome Sequencing

This method delivers a comprehensive view of genetic variation, ideal for discovery applications.

  • Advantages of High-Throughput Sequencing: Process more samples to improve statistical power, and cost-effectively run emerging data-rich methods, including single-cell and spatial analyses.
  • NGS Data Analysis: Find user-friendly tools and tips to smooth the process of analyzing sequencing data, so you can spend more time doing research and less time configuring workflows.
  • Multiomics Profiling: Combine genomic data with data from other modalities such as transcriptomics, epigenetics, and proteomics, to better connect genotype to phenotype and fuel discovery of novel drug targets.
In-Depth Guide to Targeted NGS

Learn more about how NGS-based targeted resequencing can help you identify variants in less time and for less money than Sanger sequencing.

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NGS vs. Sanger Sequencing

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Methods Guide
Methods Guide

All the information you need, from BeadChips to library preparation to sequencer selection and analysis. Select the best tools for your lab.

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Sequencing Technology Video
Sequencing Technology Video

See Illumina sequencing technology in action and learn how it works.

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Global Perspectives on the Impact of NGS
Global Perspectives on the Impact of NGS

Scientists from around the world share how NGS has revolutionized their fields, enabling studies that weren’t possible before.

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Product Selection and Experiment Planning Tools
Product Selection and Experiment Planning Tools

Find the right library prep kit or microarray, calculate sequencing coverage, explore methods, design custom assays, and more.

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References
  1. Jamuar SS, Lam AT, Kircher M, et al. Somatic mutations in cerebral cortical malformations. N Engl J Med. 2014;371(8):733-743.
  2. Rivas MA, Beaudoin M, Gardet A, et al. Deep resequencing of GWAS loci identifies independent low-frequency variants associated with inflammatory bowel disease. Nat Genet. 2011;43(11):1066-1073.
  3. König K, Peifer M, Fassunke J, et al. Implementation of amplicon parallel sequencing leads to improvement of diagnosis and therapy of lung cancer patients. J Thorac Oncol. 2015;10(7):1049-1057.
  4. Shendure J and Ji H. Next-generation DNA sequencing. Nat Biotechnol. 2008;26(10):1135-1145.
  5. Schuster SC. Next-generation sequencing transforms today’s biology. Nat Methods. 2008;5(1):16-18.