Custom-Primed Rolling Circle Amplicons for Highly Accurate Nanopore Sequencing

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Rolling circle amplification (RCA) generates tandem DNA repeats but traditional methods yield branched, short products unsuitable for nanopore sequencing.
• Nanopore sequencing offers high accuracy for repetitive DNA via consensus calling, but requires long, linear amplicons.

Purpose of the Study:

• To develop an enhanced RCA protocol for producing longer, less branched amplicons.
• To establish a highly accurate nanopore sequencing pipeline using these amplicons for tandem repeat analysis.

Main Methods:

• Developed an enhanced RCA protocol using sequence-specific primers to generate long, linear amplicons.
• Created a custom-primed rolling circle amplification sequencing (CPRSeq) pipeline leveraging these amplicons.
• Applied CPRSeq to sequence tumor-associated single nucleotide variants and perform whole-genome sequencing of E. coli.

Main Results:

• The enhanced RCA protocol successfully produced longer and less branched DNA amplicons.
• CPRSeq demonstrated high accuracy in sequencing standard samples with low-frequency tumor variants.
• Achieved successful whole-genome sequencing and assembly of E. coli using the CPRSeq pipeline.

Conclusions:

• The developed CPRSeq pipeline significantly enhances nanopore sequencing accuracy for repetitive DNA.
• CPRSeq is a powerful tool for sensitive variant detection and comprehensive genome analysis.
• This method advances genomic applications requiring precise sequencing of tandem repeats.