Sequencing Experiment Planning

Key Questions to Consider

• Which sequencing method is right for your application?
• Should you use Sanger sequencing or Next Generation Sequencing (NGS)?

Evolution of Sequencing Technologies

• Before the 1990s: Traditional methods using gels and manual base calling.
• Sanger Sequencing: Introduced capillary electrophoresis, automated base calling, capable of sequencing long DNA fragments.
• Next Generation Sequencing (NGS): Transformed sequencing with massively parallel processing, allowing for millions of fragments to be sequenced simultaneously.

Comparison of Sanger Sequencing and NGS

Sanger Sequencing

• Accuracy: 99.99% accuracy, considered gold standard for clinical research.
• Applications:
  • Sequencing single genes.
  • Sequencing 1-100 amplicon targets cost-effectively.
  • Sequencing up to 96 samples at once without barcoding.
  • Microbial identification.
  • Fragment analysis and high-throughput genotyping (e.g., SNaPshot).
  • Microsatellite or STR analysis.
  • NGS confirmation.

Next Generation Sequencing (NGS)

• Turnaround Time: Fast, around 4 hours to complete a run.
• Applications:
  • Cost-effective interrogation of >100 genes at a time.
  • Finding novel variants by expanding target sequences in a single run.
  • Sequencing samples with low input amounts of starting material (as low as 10 ng with Ion AmpliSeq library preparation).
  • Sequencing microbial genomes for pathogen subtyping in outbreak situations.

Conclusion

• Both Sanger and NGS have their strengths and are suitable for different applications.
• Sanger Sequencing: Best for high accuracy and small scale.
• NGS: Best for high throughput and cost efficiency.

Additional Information

• For questions, visit: Thermo Fisher Ask
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Remember

• When in doubt, just Seq It Out!