Complex Diseases & Personalised Medicine

Complex Diseases & Personalised Medicine

Introduction

Maria Pala's lecture discusses complex diseases and personalized medicine, covering high-throughput sequencing, pharmacogenomics, and strategies for analyzing complex diseases.

Review of Last Week

  • Human diseases can be mimicked using in vitro and in vivo models.
  • Animal models are generated through gene transfer and gene targeting.
  • Genomic engineering utilizes programmable nucleases and homologous recombination.
  • CRISPR/Cas is a flexible system adaptable for DNA and RNA engineering.

Learning Outcomes

  • Describe technologies to analyze whole human genomes in a single experiment.
  • Describe applications of personalized medicine using pharmacogenomics.
  • Understand major differences between monogenic and complex diseases and identify strategies to analyze complex diseases.

Part 1: High-Throughput Sequencing

High-Throughput Sequencing Overview
  • High-throughput sequencing involves:
    1. Genome fragmentation.
    2. Genomic selection and enrichment.
    3. DNA capture and amplification.
    4. DNA library preparation and sequencing.
DNA Immobilization
  • High throughput is achieved by immobilizing DNA target fragments.
  • A sequence library is prepared and immobilized by base pairing to attached standard oligonucleotides, contrasting with Sanger sequencing.
DNA Amplification
  • DNA is amplified by adaptor-primed PCR.
  • PCR products are immobilized by oligonucleotide binding to a glass slide.
Illumina Reversible Terminator Sequencing
  • High-throughput sequencing methods are often named after their manufacturers.
  • Illumina uses a sequencing-by-synthesis method with reversible dye terminators.
Parallel Short-Read Sequencing
  • In Illumina sequencing, DNA fragments are ligated to adapters containing unique molecular identifiers (barcodes).
  • Adapter-tagged DNA is loaded onto a flow cell, and copies are generated through bridge amplification.
  • Sequencing by synthesis incorporates fluorescently labeled dNTPs.
  • The final output is a set of recorded images, with each color representing a specific nucleotide.
Single-Molecule Sequencing
  • Single-molecule long-read sequencing uses an optical system (zero-mode waveguide, ZMW) to detect incorporated bases in real-time.
    • No PCR amplification is needed.
    • No termination step.
    • Still relies on sequencing by synthesis.
    • Advantage: Long reads.
    • Disadvantage: Mentioned, but not elaborated.
SMRT (Single-Molecule Real-Time) Sequencing
  • In PacBio SMRT sequencing, dsDNA is fragmented and ligated to hairpin adapters to form a circular SMRTbell molecule.
  • The molecule is bound by a DNA polymerase and loaded onto a SMRT Cell for sequencing.
  • Each SMRT Cell contains up to 8 million zero-mode waveguides (ZMWs).
  • Light penetrates the lower 20-30 nm of each well, reducing the detection volume to only 10−2110^{-21} L.
  • The SMRTbell template and polymerase are immobilized at the bottom, and fluorescently labeled dNTPs are added for signal detection.
SMRT Sequencing Process
  • When a fluorescent dNTP is held in the detection volume, a light pulse excites the fluorophore.
  • The emitted light is detected by a camera, recording the wavelength and position of the incorporated base.
  • The fluorophore is cleaved to prevent interference during the next pulse.
  • The DNA sequence is determined by the recorded fluorescent emission within each ZMW.
Nanopore Sequencing
  • A single, long DNA molecule passes through a nanopore in a membrane within an electrical field.
    • No PCR amplification.
    • No sequencing by synthesis.
    • Real-time sequencing.
  • The sequence is determined by the pattern of perturbed ion flow through the nanopore.
Comparison of Sequencing Technologies
  • A table compares first and next-generation DNA sequencing technologies based on:
    • Generation, data read length, accuracy, throughput per run/cell, cost per Gb, and Gb per year.
    • Includes Sanger, Illumina Reversible terminator, Thermo Semi-conductor, PacBio SMRT, and Oxford Nanopore.
    • Example values:
    • Illumina Reversible terminator: Read length = 2×0.252 \times 0.25 kb, Accuracy > 99.9%, Throughput > 65 Gb
    • PacBio SMRT: Read length > 30 kb, Accuracy = 90%, Throughput > 50 Gb
    • Oxford Nanopore: Read length > 10 kb, Accuracy = 93%, Throughput > 2 Gb

Part 2: Personalized Medicine

Relevance of Individual DNA Variation
  • High-throughput sequencing identifies thousands of variants compared to reference genomes.
    • Pathogenic variants cause monogenic diseases.
    • Variants contribute to polygenic or multifactorial disease susceptibility.
    • Variants affect drug action and side effects.
Individual Variation of Drug Responses
  • Pharmacokinetic variation: variable drug uptake, distribution, and metabolism.
  • Pharmacodynamic variation: variable responsiveness of the drug target.
  • Influenced by genetic factors, drug interactions, age, and disease.
Ethical Issues in High-Throughput DNA Sequencing
  • Incidental findings.
  • Confidentiality and privacy.
  • Neonatal screening.
  • Genetic discrimination.

- Genetic manipulation.