Lecture 5: Sequencing Technology

Objectives of Sequencing Technology

• Differentiate between Sanger sequencing and Next Generation Sequencing (NGS).
• PCR advancements have simplified sequencing processes.
• Capable of sequencing anything from short DNA fragments to entire genomes.
• Provides insights into polymorphisms, mutations, and evolutionary data.

Common Sequencing Methods

• Enzymatic addition of nucleotides complementary to the DNA template.
• Utilization of DNA polymerase for nucleotide addition.
• Identification of added bases to determine sequence information.

Sanger Sequencing

• Uses dNTPs with the addition of ddNTPs, which terminate DNA strand extension due to lack of hydroxyl group.
• Each ddNTP is tagged with a unique fluorescent dye to identify the terminating nucleotide.
• Generates short DNA fragments corresponding to each possible termination point.
• Requires separation of bands/peaks to read the DNA sequence from the shortest to the longest fragment.
• Quality issues can arise from inadequate template, poor primer design, or impure DNA.

Next Generation Sequencing (NGS)

• Includes platforms like Illumina, 454, and nanopore technologies.
• Enables whole genome, de novo, and RNA sequencing.
• Generates millions of sequences simultaneously, offering high-throughput and cost-effectiveness.

Steps in Next Generation Sequencing

• DNA Fragmentation:

  • Breaks genomic DNA into smaller fragments (200-600 base pairs) using mechanical or enzymatic methods.
  • Facilitates easier handling and analysis.

• Phosphorylation of DNA Fragments:

  • Kinase enzymes add phosphate groups to the DNA ends to enable ligation of adapters.

• Ligation of Adapters:

  • Short DNA sequences are attached to fragmented DNA, allowing for binding and amplification.
  • Contains sequences for PCR amplification and multiplexing capabilities.

• Size Selection:

  • Isolates DNA fragments within a specific size range using methods like gel electrophoresis.
  • Ensures sequencing quality by avoiding size bias.

• Fragments Capture on Oligo-Coated Slides:

  • Prepared DNA library is applied to slides coated with oligonucleotides complementary to adapters.

• DNA Polymerase Synthesis:

  • Amplifies DNA fragments through Bridge PCR, generating clusters of identical molecules for improved signal detection during sequencing.

• Addition of Sequencing Primers and Reads:

  • Sequencing primers are introduced to DNA clusters, where labeled nucleotides produce distinct fluorescent signals, allowing real-time monitoring and recording of sequences.

Genome Sequence Assembly

• Reconstructs original genomes from overlapping short DNA sequences.
• Provides redundancy for filtering sequencing errors, enhancing reliability of assembled genomes.

Examples of Sequencing Experiments and Their Uses

• Whole Genome Sequencing (WGS):

  • Sequences the complete genomic DNA, useful for identifying genetic variants and evolutionary studies.

• Exome Sequencing:

  • Focuses on exons, aiding in disease diagnosis and cancer genomics.

• RNA Sequencing (RNA-Seq):

  • Analyzes gene expression levels, alternative splicing, and non-coding RNAs.

• Targeted Sequencing:

  • Examines specific genomic regions, important in cancer genotyping and hereditary disease studies.

• Metagenomic Sequencing:

  • Sequences collective microbial genomes from environmental samples, providing comprehensive insights into genetic variation.

Disadvantages of NGS

• Generates large data volumes, requiring substantial computational resources.
• Produces shorter reads, complicating assembly in complex regions.
• Higher error rates necessitate validation of findings.
• Extensive library preparation may introduce biases.

Advantages of Sanger Sequencing

• High accuracy, considered the gold standard for variant validation.
• Longer read lengths enable sequencing of complex regions.
• Straightforward workflow requiring less computational power.
• Established and widely accepted technique in clinical and research settings.

Disadvantages of Sanger Sequencing

• Low throughput, sequencing one fragment at a time.
• Higher cost per base compared to NGS.
• Longer turnaround times, particularly for larger projects.
• Limited to specific genes or smaller regions, unsuitable for large-scale sequencing endeavors.