DNA Sequencing 3

Third Generation DNA Sequencing

Introduction

• Course: MMG2040 2026

• Key Focus: Third Generation Sequencing (TGS)

Comparison of Next Generation Sequencing (NGS) Platforms

• Features of Different Platforms:

  • Ion Torrent:

  • Detection: Measures pH change (H⁺)

  • Speed: Faster than other platforms

  • Accuracy: Lower accuracy in repetitive sequences

  • Equipment: Utilizes a semiconductor chip

  • Illumina:

  • Detection: Based on fluorescence

  • Speed: Slightly slower than Ion Torrent

  • Accuracy: Very high accuracy

  • Equipment: Employs an optical imaging system

Third Generation Sequencing (TGS) Overview

• Characteristics of TGS:

  • Single-Molecule Sequencing: Can sequence individual molecules directly without amplification.

  • No PCR Amplification Required: Avoids biases introduced by the amplification process.

  • Real-Time Sequencing: Capable of providing results as the sequencing occurs.

  • Long-Read Capabilities: Can generate reads from 10 kb to over 1 Mb.

  • Single Strand Sequencing: Only one strand of DNA is needed, eliminating the demand for multiple copies.

• Technologies:

  • Nanopore Detectors: Utilizes nanopore technology for sequencing.

  • SMRT Sequencing (Single Molecule Real-Time): A key technology used in TGS.

Reasons for Moving Beyond NGS

• Limitations of NGS:

  • Short Reads: Make genome assembly a challenge.

  • Difficulty Resolving Repeats: Short reads struggle to resolve repetitive regions effectively.

  • Limited Structural Variant Detection: Short reads are unable to identify larger structural variants.

  • PCR Bias: Amplification processes introduce biases that can affect data integrity.

Nanopore Sequencing Overview

• Process:

  • DNA or RNA passes through a nanopore protein, which measures changes in ionic current.

  • Changes in the ionic current are translated into a nucleotide sequence.

• Development: Developed by Oxford Nanopore Technologies.

Nanopore Detectors in TGS

• Process:

  • Single-stranded DNA (ssDNA) passes through a nanopore approximately 1 nm in diameter.

  • Each base of the DNA sequence generates a unique signal change in the electrical current.

  • Error-Prone: The process is considered to be error-prone.

  • 1X Coverage: Generally provides one coverage depth, which is acceptable for quick analyses or re-sequencing.

How Nanopore Sequencing Works

• Mechanism:

  • A motor protein unwinds the DNA double helix.

  • The single strand of DNA passes through the nanopore.

  • Each nucleotide base alters the electrical current, and machine learning algorithms decode these signals into nucleotide sequences.

Advantages and Disadvantages of Nanopore Sequencing

• Advantages:

  • Capable of ultra-long reads (greater than 1 Mb).

  • Portable devices available (e.g., MinION).

  • Permits real-time sequencing and the capability for direct RNA sequencing.

  • Lower capital costs compared to some other sequencing methods.

• Disadvantages:

  • Higher error rates, averaging around 5% to 10%.

  • Presence of signal noise can complicate data interpretation.

  • Complexity of data analysis can be a challenge to many users.

  • Lower throughput compared to Illumina platforms.

PacBio Sequencing Overview

• Technology: Single Molecule Real-Time (SMRT) sequencing.

• Components:

  • Utilizes DNA polymerase combined with zero-mode waveguides (ZMWs).

  • Employs fluorescently labeled nucleotides for detection.

Detailed Features of PacBio Sequencing

• Process:

  • Single Molecule Real-Time (SMRT): Also known as PacBio.

  • Nanocontainers: ZMWs facilitate the single DNA template fitting, with each container having a diameter of 20 nm.

  • Detection of Incorporation: Fluorescent deoxynucleotide triphosphates (dNTPs) report the incorporation of nucleotides, which results in a distinctive light flash as the pyrophosphate group is discarded.

  • Typical Read Length: About 20,000 base pairs (bps). Works effectively for sequencing repetitive sequences.

How PacBio Sequencing Works

• Mechanism:

  • DNA polymerase is anchored in the ZMW and incorporates nucleotides while emitting light.

  • Each base emits a distinct fluorescence signal that is detected in real-time, enabling continuous monitoring of DNA mutations or variations as they occur.

Advantages and Disadvantages of PacBio Sequencing

• Advantages:

  • Long Read Lengths: Typical reads range from 10 kb to 100 kb.

  • High Consensus Accuracy: Known as HiFi reads, offering improved accuracy.

  • No Amplification Bias: Eliminates biases since no PCR is utilized.

  • Detection of Epigenetic Modifications: Capable of identifying epigenetic changes in DNA.

• Disadvantages:

  • Generally higher costs compared to nanopore sequencing.

  • Lower throughput compared to Illumina sequencing methodologies.

  • Requires specialized equipment for operation.

Comparison of PacBio and Other NGS Platforms

• Features Comparison:

  • Read Length: PacBio (very long) vs. Illumina/Ion Torrent (short).

  • Amplification: PacBio (none) vs. Illumina/Ion Torrent (Yes).

  • Detection Method: PacBio uses fluorescence (real-time) while Illumina and Ion Torrent use fluorescence (stepwise) and pH, respectively.

• Strengths:

  • Genome Assembly: PacBio excels in assembling complex genomes due to longer reads.

  • Accuracy: Illumina offers high throughput while PacBio emphasizes accuracy and read length.

Applications of Third Generation Sequencing

• Typical applications include:

  • De Novo Genome Assembly: Constructing genomes from scratch without reference sequences.

  • Structural Variant Detection: Identifying structural variations within genomes such as insertions, deletions, and other alterations.

  • Metagenomics: Studying genetic material recovered directly from environmental samples.

  • Transcript Isoform Analysis: Analyzing different versions of RNA transcripts from a single gene.

  • Epigenetics: Studying the heritable changes in gene expression that do not involve changes to the underlying DNA sequence.

Comparison of Sanger, NGS, and Third Generation Sequencing

• Sanger Sequencing: Known for low throughput but high accuracy with short reads.

• Next Generation Sequencing (NGS): Offers high throughput but produces short reads (e.g., Illumina).

• Third Generation Sequencing (TGS): Delivers long reads, real-time capabilities, and the ability to work with single molecules directly.

Key Differences Among Sequencing Methods

• Read Length:

  • Shortest: Sanger < NGS < TGS (third Generation Sequencing).

• Accuracy:

  • Highest: Sanger, with improvements observed in TGS methods.

• Throughput:

  • Highest: NGS platforms.

• Cost per Base:

  • Lowest: NGS platforms, typically more affordable than both PacBio and nanopore technologies.

When to Use Each Sequencing Method

• Usage Guidelines:

  • Sanger Sequencing: Best suited for validation of small regions or specific applications requiring very high accuracy.

  • NGS: Ideal for large-scale sequencing projects, including RNA sequencing (RNA-seq).

  • Third Generation Sequencing: Highly effective for genome assembly tasks and structural variant analyses due to its long-read capabilities.

Summary of Key Points on Third Generation Sequencing

• Evolving technology enabling long-read sequencing capacity.

• Nanopore Sequencing: Notable for its portability, flexibility, and ability for real-time results.

• PacBio Sequencing: Recognized for its high accuracy and ability to produce long reads.

• Serves as a complementary technology to existing NGS platforms, enhancing the completeness and accuracy of genomic projects.

Learning Objectives

• After reviewing the content, students should be able to:

  • Describe fundamental steps in various sequencing technologies: Illumina, Ion Torrent, Nanopore, and SMRT sequencing.

  • Understand the principles governing third generation sequencing methodologies.

  • Describe specific technologies, including Nanopore and PacBio, and how they differ from Sanger/NGS methods.

  • Evaluate applications, advantages, and limitations associated with each sequencing platform.