A simple and rapid library preparation for Oxford nanopore amplicon sequencing using reverse complement PCR

Area of Science:

• Molecular Biology
• Genomics

Background:

• Oxford Nanopore sequencing offers rapid and scalable amplicon sequencing.
• Conventional library preparation for multiplexed samples is time-consuming and labor-intensive.

Purpose of the Study:

• To develop a simplified and rapid library preparation method for Oxford Nanopore amplicon sequencing.
• To reduce hands-on time and post-PCR processing for multiplexed samples.

Main Methods:

• Combined reverse complement PCR (RC-PCR) with Rapid Barcode Primers.
• Incorporated multiplexing barcodes and ligase-free adapter attachment during amplification.
• Utilized a single solid-phase reversible immobilization clean-up and a five-minute adapter attachment step.

Main Results:

• Achieved successful amplification at optimized reverse-complement probe concentrations.
• Substantially reduced hands-on time compared to standard protocols.
• Preserved full-length amplicons, demonstrating method efficacy.

Conclusions:

• The developed protocol offers a rapid and efficient alternative for nanopore amplicon sequencing.
• This method is particularly suitable for time-sensitive applications requiring fast turnaround.
• Simplifies library preparation for multiplexed samples in nanopore sequencing workflows.