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 high-throughput 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 throughput 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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  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 TruSeq Custom Amplicon workflow)

Options for Sanger vs. Next-Generation Sequencing

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.

Cost-Effectiveness of Targeted NGS vs. Sanger Sequencing
Benefits of Targeted Sequencing

Learn how targeted NGS can help you gain greater insights, save time, and be more confident in your results compared to Sanger sequencing and other traditional methods.

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Benefits of Targeted Sequencing
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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Profiling Genetic Variants to Understand Lipid Disorders

The Robarts Research Institute saved time, costs, and labor by transitioning its clinical research from Sanger sequencing to NGS.

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Targeted Resequencing
Targeted Resequencing

With targeted resequencing, a subset of genes or a genomic region is isolated and sequenced, which can conserve lab resources. Learn more about targeted resequencing.

Whole-Genome Sequencing
Advantages of Whole-Genome Sequencing

Whole-genome sequencing delivers a comprehensive view of genetic variation, ideal for discovery applications. Learn more about whole-genome sequencing.

NGS vs. Sanger Sequencing

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NGS for Rare Mutation Detection
NGS for Rare Mutation Detection

While Sanger sequencing was unable to detect rare variants, NGS could identify mosaicism down to a 1% limit of detection.

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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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In-Depth Introduction to NGS
In-Depth Introduction to NGS

This detailed overview describes major advances in technology, the basics of Illumina sequencing chemistry, and more.

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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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Simple NGS Data Analysis for Biologists
Simple NGS Data Analysis for Biologists

An Oxford lab uses BaseSpace Sequence Hub to manage data analysis, without needing an experienced bioinformatician.

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Benefits of BaseSpace Sequence Hub
Benefits of BaseSpace Sequence Hub

Our informatics platform allows researchers to set up and monitor runs, analyze data, and share with collaborators easily.

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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.