Human Genome Project and Next Generation Sequencing

Human Genome Project

• First major sequencing project
• Used Sanger Sequencing
• Completed in 2003
• Took 13 years to complete
• Cost 3 Billion dollars

Next Generation Sequencing

• Introduced in 2004 and 2006
• Offers high-throughput sequencing
• Massively parallel sequencing technology
• Allows sequencing of entire genomes or regions of interest
• Reduced cost per megabase
• Increased speed and accuracy
• Allows for a wide variety of applications

Next Generation Sequencing Applications

• Rapidly sequence whole genomes
• Deeply sequence target regions
• Utilize RNA sequencing (RNA-seq) to discover novel RNA variants and splice sites
• Quantify mRNAs for gene expression analysis
• Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions
• Sequence cancer samples to study rare somatic variants and tumour subclones
• Identify novel pathogens

Categories of NGS

• Two categories
  • Sequencing by hybridization
    • Uses probes to interrogate sequences
    • Used in diagnostic applications for identifying:
      • Disease-related SNPs
      • Gross chromosome abnormalities – rearrangements, deletions, duplications, copy number variants (CNVs)
  • Sequencing by synthesis (SBS)
    • A development of Sanger sequencing (but without ddNTPs)
    • Uses repeated synthesis cycles and methods to incorporate nucleotides into a growing chain

Similarities Between Different NGS Technologies

• Sample preparation

  • Amplification or ligation with custom adapter sequences
  • Adapter sequences allow library hybridization to the sequencing chips
  • Universal priming site for sequencing primers

• Sequencing machines

  • Library fragments are amplified on a solid surface – beads or flat silicon
  • Amplification creates clusters of DNA, each originating from a single library fragment
  • Sequence from each cluster is optically read - by light or fluorescence

• Data output

  • Each machine provides raw data at the end of the sequencing run
  • This data is analyzed to provide meaningful results

Sequencing by Synthesis Properties

• Relies on shorter reads (300-500bp)
• Intrinsically higher error rate relative to Sanger sequencing
• Requires high sequence coverage – "massively parallel sequencing"
• Utilizes step-by-step incorporation of reversibly fluorescent and terminated nucleotides

Illumina Sequencing - Library Preparation

• DNA is broken into manageable fragments (~200-600bp)
• Adapters are attached to DNA fragments
• DNA fragments attached to adapters are denatured
• Libraries are constructed to give a mixture of adapter-flanked fragments

Illumina Sequencing - Bridge Amplification

• Utilizes bridge PCR to amplify the genomic region being sequenced
• Primers densely coat the surface of a solid substrate
• Amplification products from the template library remain locally attached near the point of origin
• Each clonal cluster contains ~1000 copies of a single member of the template library
• DNA fragments are made single-stranded
• DNA fragments are washed across the flow cell
• Complementary DNA binds to primers on the surface of the flow cell
• DNA that doesn’t attach is washed away

Illumina Sequencing – Bridge Amplification - Clonal Clustering

• Double-stranded clonal bridges are denatured
• Reverse strands are removed
• Forward strands remain as clusters for sequencing

Illumina Sequencing – Sequencing Reaction

• Cyclic reversible termination
• One of the four fluorescent dNTPs is added per cycle
• Images of clusters are captured after incorporation of each nucleotide
• After imaging, the dye and terminator are cleaved and released

When To Use NGS vs Sanger Sequencing

• Sanger Sequencing
  • Suitable for small regions of DNA or limited number of samples
• NGS
  • Cost-effective for screening multiple samples
  • Allows detection of multiple variants across targeted areas of the genome
  • More cost-effective and time-saving when Sanger sequencing would be too costly or time-consuming

Explore the key milestones of the Human Genome Project and the advancements brought by Next Generation Sequencing (NGS). Discover the technologies, applications, and categories of NGS that have revolutionized genomic research since the early 2000s. This quiz will challenge your understanding of these pivotal developments in genetics.