Introduction to Nanopore Sequencing

Brief History

Nanopore sequencing is a relatively recent advancement in the field of DNA sequencing. The foundational concept was introduced in the 1990s, with key progress made by researchers exploring the passage of DNA through biological pores. Oxford Nanopore Technologies (ONT), founded in 2005, commercialized the technology, launching its first portable sequencer, the MinION, in 2014. This innovation made real-time, long-read sequencing accessible and scalable.

How Nanopore Sequencing Works

Nanopore sequencing uses a simple but powerful principle: as single-stranded DNA (or RNA) passes through a nanopore (a nanometer-scale hole), it causes characteristic disruptions in an ionic current. These disruptions correspond to specific nucleotide sequences.

Core Components:

• Nanopore: A biological or synthetic pore embedded in a membrane.
• Motor Protein: Controls the rate at which nucleic acids pass through the pore.
• Electrolyte and Voltage: An applied voltage drives the DNA or RNA through the pore, generating a measurable ionic current.
• Signal Detection: Each base (A, T, G, C) alters the current in a unique way. Software decodes this signal into a nucleotide sequence ("basecalling").

Advantages:

• Long read lengths (up to megabases)
• Real-time sequencing
• Minimal sample prep
• Portable and scalable (e.g., MinION, GridION, PromethION)

Applications in Plasmid and Genome Sequencing

Plasmid Sequencing:

Nanopore sequencing is ideal for plasmids because:

• It can sequence entire plasmids in a single read (de novo assembly)
• Detects base modifications (e.g., methylation)
• Fast turnaround for QC and verification

Typical Workflow:

1. Isolate plasmid DNA
2. Prepare library using a rapid or ligation kit
3. Load onto a MinION flow cell
4. Sequence and assemble using tools like Flye or Miniasm

Whole Genome Sequencing (WGS):

Nanopore is powerful for microbial and even eukaryotic WGS:

• Captures structural variants, repeats, and large rearrangements
• Used for hybrid assemblies with short-read data (e.g., Illumina)
• Supports metagenomics and direct pathogen sequencing

Workflow:

1. Extract high molecular weight (HMW) DNA
2. Library prep with ligation sequencing kit
3. Run on MinION or higher-throughput device
4. Use tools like Canu, Flye, or Medaka for assembly and polishing

Comparison Table: Nanopore vs. PacBio vs. Illumina

Feature	Nanopore (ONT)	PacBio HiFi	Illumina (Short-read)
Read Length	Up to >2 Mb	~10–25 kb (HiFi reads)	150–300 bp
Accuracy (raw/base-called)	~95% (raw), >99% (polished)	~99.9%	>99.9%
Instrument Cost	Low (MinION) to high	High	High

Summary

Nanopore sequencing is revolutionizing genomics with its real-time, portable, and long-read capabilities. It is particularly effective for plasmid sequencing and structural variant detection in genomes. While not as accurate as PacBio or Illumina out of the box, nanopore data can be polished to high quality, and its unique advantages make it a powerful tool for modern genomics workflows.