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Lecture 02A
DNA sequencing

Foundations

Date: Jan 14, 2024

This session provides the conceptual framework for understanding modern DNA sequencing technologies and the importance of quality control in genomic data analysis. Students will explore the principles underlying sequencing techniques and file formats.

Learning objectives

After today, you should have a better understanding of:

  1. Importance and applications of DNA sequencing.
  2. Techniques for extracting and purifying high-quality DNA.
  3. Steps in preparing DNA libraries for sequencing.
  4. Principles and innovations of DNA sequencing technologies.

Outline

Importance and Applications of DNA Sequencing

By determining the precise order of nucleotides in DNA, sequencing technologies enable breakthroughs in understanding genetic information, driving innovations in medicine, agriculture, biotechnology, environmental science, and beyond.

  • Understand how DNA sequencing has revolutionized biological research and genomics.
  • Describe the use of DNA sequencing in healthcare, agriculture, biotechnology, environmental monitoring, and forensics.

DNA Extraction and Purification

Establish a foundational understanding of the techniques and methodologies in extracting and purifying DNA from biological samples, ensuring high-quality input for subsequent sequencing processes.

  • Describe the various methods for DNA extraction, including enzymatic, mechanical, and chemical approaches.
  • Explain the processes of purifying extracted DNA to remove contaminants and inhibitors.
  • Demonstrate techniques for assessing the purity and concentration of extracted DNA (e.g., spectrophotometry, fluorometry).

DNA Library Preparation for Sequencing

Understand the critical steps in preparing DNA libraries for sequencing, including fragmentation, adapter ligation, and indexing, to ensure compatibility with various sequencing platforms.

  • Explain the methods used to fragment DNA into suitable sizes for sequencing (e.g., mechanical shearing, enzymatic digestion).
  • Describe the process of attaching sequencing adapters to fragmented DNA and the importance of adapter design.
  • Discuss the role of indexing in allowing multiple samples to be sequenced simultaneously and the methods used for indexing.
  • Identify key quality control metrics to assess library quality before sequencing (e.g., library size distribution, concentration).

Scientific Principles of DNA Sequencing Technologies

Explore the historical development and underlying scientific principles of various DNA sequencing technologies, highlighting key innovations and their impacts on the field.

  • Outline the evolution of DNA sequencing methods from Sanger sequencing to next-generation (NGS) and third-generation technologies.
  • Explain the scientific principles behind Sanger sequencing, including chain termination and fluorescence detection.
  • Describe the core principles of sequencing by synthesis, focusing on reversible terminators and bridge amplification.
  • Explain how single-molecule sequencing platforms detect and interpret DNA bases.
  • Compare the fundamental advantages and challenges of second- and third-generation sequencing methods.

Supplementary material

Relevant content for today's lecture.

Presentation