Highly accurate single-color fluorogenic DNA decoding sequencing for mutational genotyping

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Current DNA sequencing technologies face limitations in accuracy, read length, and throughput.
• Reducing sequencing costs and error rates is crucial for broader applications in research and clinical settings.

Purpose of the Study:

• To develop a single-color fluorogenic DNA decoding sequencing method.
• To improve sequencing accuracy, read length, throughput, and reduce scanning time.
• To enable efficient detection and correction of sequencing errors and mutation sites.

Main Methods:

• Incorporation of four types of 3'-O-modified nucleotide reversible terminators, with two labeled by the same fluorophore and two unlabeled.
• Cyclic interrogation of DNA templates twice with different nucleotide combinations to obtain sequential encodings.
• Demonstration of feasibility using established sequencing chemistry and validation on human mitochondrial DNA.

Main Results:

• Achieved a cycle efficiency of approximately 99.5%.
• Demonstrated a theoretical error rate of 0.00016% at a sequencing depth of ×2, outperforming Sanger sequencing.
• Successfully detected a mutation site in human mitochondrial DNA from a single sequencing run.

Conclusions:

• The proposed single-color fluorogenic DNA sequencing method offers significant improvements in accuracy, read length, and efficiency.
• The method is compatible with existing sequencing-by-synthesis platforms and has the potential to reduce costs.
• This approach holds promise for advancing applications in various fields of biology and medicine.