Direct oligonucleotide sequencing with nanopores

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Third-generation DNA sequencing offers direct analysis of nucleic acids.
• Sequencing single-stranded DNA (ssDNA) or RNA provides unique insights but faces adapter compatibility challenges.
• The Oxford Nanopore MinION platform sequences single DNA strands but typically requires double-stranded DNA (dsDNA) library preparation.

Purpose of the Study:

• To develop a direct sequencing method for short, single-stranded oligonucleotides using the MinION platform.
• To enable sequencing without amplification or second-strand synthesis.
• To explore applications in DNA data storage.

Main Methods:

• A novel library preparation method involving a single annealing step for 5' phosphorylated oligonucleotides.
• Designing adapter sequences to bind the 5' end of oligonucleotides, creating a 3' adenosine overhang.
• Utilizing the MinION platform for direct nanopore sequencing of prepared oligonucleotides.

Main Results:

• Successful direct sequencing of short, single-stranded oligonucleotides without amplification or second-strand synthesis.
• Recovery of approximately 90% of 120 nt orthogonal sequences from a 42,000-sequence pool with high accuracy.
• Identification of potential artifacts in nanopore raw data, including empty signals and fused sequences.

Conclusions:

• This direct oligonucleotide sequencing method is compatible with ligation-based library preparation for nanopore sequencing.
• The technique overcomes adapter compatibility issues for single-stranded nucleic acids.
• Enables novel applications, particularly in DNA data storage systems utilizing oligonucleotides as information carriers.