DNA Sequencing: Human Genome and NGS Overview
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Questions and Answers

What is formed during bridge amplification in Illumina sequencing?

  • Triple-stranded DNA structures
  • Linear DNA molecules
  • Double-stranded clonal bridges (correct)
  • Single-stranded DNA fragments
  • Which component is NOT involved in the sequencing reaction of Illumina sequencing?

  • Primers
  • Reverse strands (correct)
  • DNA polymerase
  • dNTPs labeled with a fluorescent dye
  • What is the purpose of the reversible terminator in Illumina sequencing reactions?

  • To prevent the denaturation of DNA strands
  • To ensure only one dNTP is incorporated in each cycle (correct)
  • To facilitate the removal of fluorescent dye after imaging
  • To allow the incorporation of all four dNTPs simultaneously
  • In what scenario is Sanger sequencing more preferable than NGS?

    <p>When examining a small region of DNA with limited samples</p> Signup and view all the answers

    What happens to the fluorescent dye and terminator after imaging in Illumina sequencing?

    <p>They are cleaved and released</p> Signup and view all the answers

    What is the purpose of the reversible termination in the nucleotide incorporation process?

    <p>To allow only one nucleotide to be added per cycle</p> Signup and view all the answers

    Which step involves breaking DNA into manageable fragments for Illumina sequencing?

    <p>DNA fragmentation</p> Signup and view all the answers

    What feature is characteristic of the bridge PCR in Illumina sequencing?

    <p>Primers are fixed in place on a solid substrate</p> Signup and view all the answers

    Which NGS technology uses pyrosequencing?

    <p>454 sequencing</p> Signup and view all the answers

    What happens to the DNA fragments that do not attach to the primers during the bridge amplification?

    <p>They are washed away from the flow cell</p> Signup and view all the answers

    During the sequencing by synthesis process, what occurs after the incorporation of a single nucleotide?

    <p>Remaining nucleotides are washed away</p> Signup and view all the answers

    What is the primary purpose of library quantitation in Illumina sequencing?

    <p>To determine the concentration of DNA templates</p> Signup and view all the answers

    In which step of Illumina sequencing are short sequences of DNA adapters attached to the fragments?

    <p>Adaptor ligation</p> Signup and view all the answers

    What was the duration and cost of the Human Genome Project?

    <p>13 years and $3 billion</p> Signup and view all the answers

    What are the two major categories of Next Generation Sequencing (NGS)?

    <p>Sequencing by Hybridization and Sequencing by Synthesis</p> Signup and view all the answers

    What major advantage does Next Generation Sequencing (NGS) offer over traditional sequencing methods?

    <p>Ultra-high throughput and scalability</p> Signup and view all the answers

    How has Next Generation Sequencing (NGS) impacted the cost of sequencing?

    <p>Decreased the cost per megabase</p> Signup and view all the answers

    What application does RNA sequencing (RNA-seq) NOT typically assist with?

    <p>Analyse genome-wide DNA methylation</p> Signup and view all the answers

    What technology is NOT a major method of NGS?

    <p>Mass Spectrometry Sequencing</p> Signup and view all the answers

    What role does sequencing by hybridization play in NGS?

    <p>Diagnosing disease-related SNPs and abnormalities</p> Signup and view all the answers

    Which of the following is a key reason NGS has revolutionized biological sciences?

    <p>It allows for a greater number and diversity of sequenced genomes.</p> Signup and view all the answers

    What is the function of adapter sequences in sample preparation?

    <p>To provide a universal priming site for sequencing primers.</p> Signup and view all the answers

    How is amplification achieved on sequencing machines?

    <p>Using covalently attached DNA linkers to hybridize library adapters.</p> Signup and view all the answers

    What does raw data from a sequencing run consist of?

    <p>A collection of DNA sequences generated at each cluster.</p> Signup and view all the answers

    Which characteristic is NOT typical of Sequencing by Synthesis (SBS)?

    <p>Utilizes longer sequence reads (500-700 bp).</p> Signup and view all the answers

    What is meant by 'massively parallel sequencing' in the context of SBS?

    <p>It indicates the concurrent sequencing of millions to billions of short DNA reads.</p> Signup and view all the answers

    What is a significant drawback of Sequencing by Synthesis with respect to error rates?

    <p>Incomplete removal of fluorescent signals can lead to higher background noise levels.</p> Signup and view all the answers

    What does reversibly terminating nucleotides allow in the SBS process?

    <p>Stepwise incorporation of nucleotides for accurate sequencing.</p> Signup and view all the answers

    Why is higher sequence coverage desired in Sequencing by Synthesis?

    <p>To increase the chances of detecting rare mutations.</p> Signup and view all the answers

    Study Notes

    The Human Genome Project

    • The Human Genome Project was the first major endeavor in DNA sequencing.
    • It utilized first-generation sequencing, specifically Sanger Sequencing (Chain Termination method).
    • The project lasted 13 years and cost $3 billion.
    • It was completed in 2003.

    Next Generation Sequencing (NGS)

    • NGS is the next advancement in DNA sequencing technology.
    • It is also known as Second-Generation Sequencing.
    • It was introduced in 2004 and 2006.
    • NGS offers high-throughput sequencing, meaning it can sequence many DNA fragments simultaneously.

    Advantages of NGS

    • NGS is a massively parallel sequencing technology.
    • It provides ultra-high throughput, scalability, and speed.
    • NGS is used to determine the order of nucleotides in:
      • Entire genomes
      • Targeted regions of DNA or RNA

    NGS Impact

    • NGS has revolutionized biological sciences, enabling various applications.
    • It has significantly advanced genome sequencing due to its increased speed and accuracy.
    • This leads to lower costs and reduced manpower requirements.
    • NGS has decreased the cost per megabase of sequenced DNA.
    • The number and variety of sequenced genomes have significantly increased.

    NGS Applications

    • Rapid sequencing of whole genomes
    • Deep sequencing of target regions
    • RNA sequencing (RNA-seq) to:
      • Discover novel RNA variants and splice sites
      • Quantify mRNAs for gene expression analysis
    • Analyze epigenetic factors like:
      • Genome-wide DNA methylation
      • DNA-protein interactions
    • Sequence cancer samples to study:
      • Rare somatic variants
      • Tumor subclones
    • Identify novel pathogens

    Categories of NGS

    • Two main categories:
      • Sequencing by Hybridization
        • Utilizes specific probes to interrogate sequences.
        • Used in diagnostic applications for:
          • Identifying disease-related SNPs
          • Identifying gross chromosome abnormalities like rearrangements, deletions, duplications, and copy number variants (CNVs)
      • Sequencing by Synthesis (SBS)
        • An advancement of Sanger sequencing without ddNTPs.
        • Uses repeated synthesis cycles and methods to incorporate nucleotides into a growing chain.

    Similarities Between NGS Technologies

    • Sample Preparation:
      • Requires a library obtained through amplification or ligation with custom adapter sequences.
      • Adapter sequences enable library hybridization to sequencing chips.
      • They provide universal priming sites for sequencing primers.
    • Sequencing Machines:
      • Each library fragment is amplified on a solid surface (beads or flat silicon).
      • This is done using covalently attached DNA linkers that hybridize library adapters.
      • Amplification creates DNA clusters, each originating from a single library fragment.
      • Each cluster acts as an individual sequencing reaction.
      • The sequence from each cluster is read optically using light or fluorescence.
      • Each machine has its specific cycling conditions.
    • Data Output:
      • Each machine provides raw data at the end of the sequencing run.
      • Raw data is a collection of DNA sequences generated at each cluster.
      • This data is further analyzed to provide meaningful results.

    Sequencing by Synthesis (SBS) Properties

    • Relies on shorter reads (300-500 bp).
    • Generally has a higher error rate compared to Sanger sequencing.
      • This is due to incomplete removal of fluorescent signals, leading to increased background noise.
    • Relies on high sequence coverage, known as "massively parallel sequencing."
      • This involves sequencing millions to billions of short DNA sequence reads (50-300 nucleotides).
      • This is also referred to as Short Read Sequencing.
    • Utilizes step-by-step incorporation of reversibly fluorescent and terminated nucleotides.
      • Nucleotides are modified in two ways:
        • Each nucleotide is reversibly attached to a single fluorescent molecule with unique emission wavelengths.
        • Each nucleotide is also reversibly terminated, ensuring only one nucleotide is incorporated per cycle.

    Sequencing by Synthesis (SBS) Process

    • All four nucleotides are added to the sequencing chip.
    • A single nucleotide is incorporated into the sequence.
    • Remaining nucleotides are washed away.
    • The fluorescent signal is read at each cluster and recorded.
    • Both the fluorescent molecule and terminator group are cleaved and washed away.
    • The process repeats until sequencing is complete.

    NGS Technologies

    • 454 sequencing or pyrosequencing (Roche Applied Science)
    • Solexa Technology (Used in Illumina genome analyzer)
    • The SOLiD platform (Applied biosystems)
    • Ion Torrent: Proton/PGM sequencing
    • The HeliScope Single Molecule Sequencer Technology
    • SMRT Pacific Biosciences

    Illumina (Solexa) Sequencing

    • Overview (Cyclic Reversible Termination)
    • Shin et al. 2014

    Illumina Sequencing – Step 1: Library Preparation

    • DNA is broken into smaller, manageable fragments (~200-600 bp).
    • Short sequences of DNA called adapters are attached to the DNA fragments.
    • DNA fragments with adapters are denatured (made single-stranded).
    • Libraries are constructed containing a mixture of adapter-flanked fragments up to several hundred bp in length.

    Illumina Sequencing – Step 1: Library Preparation Summary

    • DNA fragmentation
    • Adaptor ligation
    • Library quantitation

    Illumina Sequencing – Step 2: Bridge Amplification

    • Illumina technology relies on bridge PCR to amplify the genomic region for sequencing.
    • An in vitro constructed adapter-flanked library is PCR amplified.
      • Both primers densely coat the surface of a solid substrate, attached at their 5' end by a flexible linker.
      • This ensures that the amplification products remain locally attached near the point of origin.
    • At the end of PCR, each clonal cluster contains ~1000 copies of a single member of the template library.
    • DNA fragments with adapters are made single-stranded.
    • Prepared DNA fragments are washed across the flow cell (sequencing chip).
    • Complementary DNA binds to primers on the flow cell surface.
    • Unbound DNA is washed away.

    Illumina Sequencing – Step 2: Bridge Amplification

    • The complementary strand of the DNA fragment in the library is synthesized.
    • The complementary strand folds over and anneals with the other type of flow cell oligo, forming a bridge.
    • The double-stranded bridge is denatured, creating two single strands attached to the flow cell.
    • The process of bridge amplification repeats, forming more clones of double-stranded bridges.

    Illumina Sequencing – Step 2: Bridge Amplification - Clonal Clustering

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

    Illumina Sequencing – Step 3: Sequencing Reaction

    • The components are:
      • Primers
      • dNTPs
        • Labeled with a fluorescent dye.
        • Contain a reversible terminator (Trinitrogen = N3)
      • DNA polymerase

    Illumina Sequencing – Step 3: Sequencing Reaction

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

    Illumina Sequencing – Step 3

    • The sequencing reaction uses fluorescent nucleotides with a reversible terminator group, Trinitrogen (N3).

    When to Use NGS vs Sanger Sequencing

    • Sanger sequencing:
      • An appropriate choice for investigating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer).
    • NGS:
      • Allows cost-effective screening of more samples.
      • Enables the detection of multiple variants across targeted areas of the genome.
      • Ideal when Sanger sequencing approaches would be too expensive and time-consuming.

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    Description

    This quiz explores the evolution of DNA sequencing from the Human Genome Project to Next Generation Sequencing (NGS). It covers the methods, advantages, and impact of these technologies on biological sciences. Test your knowledge on key developments and innovations in DNA sequencing.

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