What is Long Read Sequencing using ONT (Oxford Nanopore)?

Long Read Sequencing using Oxford Nanopore Technologies (ONT) is a cutting-edge method of DNA and RNA sequencing that enables real-time, single-molecule, ultra-long read sequencing. ONT’s hallmark innovation is its nanopore-based technology, which detects changes in electrical current as nucleic acids pass through tiny protein nanopores embedded in membranes.

ONT sequencing allows researchers to sequence molecules thousands to even millions of bases long, making it ideal for structural variant analysis, full-length transcriptomics, metagenomics, and more.

Overview

• Platform: Oxford Nanopore Technologies (MinION, GridION, PromethION, Flongle)

• Technology: Nanopore-based real-time single-molecule sequencing

• Read Length: 10 kb to 2 Mb+ (ultra-long reads possible)

• Key Strengths: Portability, ultra-long reads, native DNA/RNA sequencing, real-time analysis

How Oxford Nanopore Sequencing Works

1. Library Preparation
   DNA or RNA is extracted and processed using ligation- or rapid-prep kits. Adapters with motor proteins are added.

2. Nanopore Sequencing
   Molecules are passed through protein nanopores embedded in a synthetic membrane. As each nucleotide moves through the pore, it disrupts the ionic current in a unique way.

3. Signal Detection
   Changes in electrical current are recorded in real-time and interpreted using basecalling software to determine the nucleotide sequence.

4. Real-Time Analysis
   Sequence data is streamed live, enabling on-the-fly decisions and dynamic experimental control.

Key Features of ONT Long Read Sequencing

Applications of Nanopore Long Read Sequencing

• De Novo Genome Assembly
  Long reads help build highly contiguous assemblies, even for complex genomes.

• Structural Variant Detection
  Easily identifies insertions, deletions, inversions, translocations, and duplications.

• Full-Length RNA Sequencing (Direct RNA-Seq or cDNA)
  Captures transcript isoforms, poly-A tails, and RNA modifications.

• Epigenetic Analysis
  Detects methylation and other DNA/RNA modifications directly from signal data.

• Metagenomics
  Enables identification of microbes and functional genes in complex environments.

• Clinical & Field Diagnostics
  Enables portable sequencing of pathogens in real-time for outbreak surveillance.

Advantages of ONT Long Read Sequencing

• Read Length Flexibility
  Sequence any fragment length — no theoretical upper limit

• Portability
  Devices like MinION and Flongle allow sequencing outside traditional labs

• Real-Time Sequencing & Analysis
  Start analyzing data within minutes of starting a run

• Direct RNA Sequencing
  Avoids bias introduced by cDNA synthesis and PCR

• Rapid Turnaround
  End-to-end workflows can deliver answers in hours

• Cost-Efficient Scaling
  Flongle (small), MinION (moderate), GridION & PromethION (high-throughput)

Limitations and Considerations

• Lower Per-Read Accuracy (Raw)
  Raw accuracy typically ~90–95%, though improved with basecalling and polishing tools

• Signal Noise
  Electrical signal detection is sensitive to conditions and requires good quality input

• Library Prep Challenges
  Ultra-long read libraries require careful handling of high molecular weight DNA

• Higher Error Rates for Homopolymers
  Sequence regions with long repeats can be error-prone without polishing

• Computational Demand
  Basecalling and data processing require substantial computing resources

Popular Tools for ONT Long-Read Data Analysis

• Guppy / Bonito – ONT’s basecalling software

• MinKNOW – Software to control the sequencer and monitor runs

• Nanopolish – Signal-level analysis and error correction

• Medaka – Neural-network-based polishing for ONT data

• Flye / Raven – Long-read genome assembly tools

• Porechop – Adapter trimming

• Filtlong – Quality filtering and read trimming

• Longshot – Variant caller for ONT data

• EPI2ME – Cloud-based real-time analysis tools from ONT