DNA Sequencing Techniques
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Questions and Answers

What is the primary purpose of DNA sequencing?

  • To amplify DNA for cloning
  • To synthesize new DNA strands
  • To determine the order of bases in a length of DNA (correct)
  • To repair mutated DNA

    • Which of the following describes Sanger sequencing?

  • A method using chain termination with ddNTPs (correct)
  • A first-generation sequencing method using multiple primers
  • A method that does not require DNA amplification
  • A method that involves high-throughput sequencing

    • What are the roles of ddNTPs in the Sanger sequencing process?

  • They provide energy for DNA synthesis
  • They act as a high-fidelity polymerase
  • They terminate DNA chain extension when incorporated (correct)
  • They introduce mutations in the DNA sequence

    • Which statement is true regarding the Sanger sequencing amplification process?

    <p>PCR amplifies the DNA to be sequenced.</p> Signup and view all the answers

    What important advancement in genomics is Frederick Sanger recognized for?

    <p>Sequencing the first whole genome</p> Signup and view all the answers

    Which is NOT a characteristic of dideoxytriphosphates (ddNTPs)?

    <p>They allow for further DNA extension once incorporated.</p> Signup and view all the answers

    Which process follows the denaturation of DNA in Sanger sequencing?

    <p>Synthesis of complementary DNA from the templates.</p> Signup and view all the answers

    Single Nucleotide Polymorphisms (SNPs) are significant in DNA sequencing because they allow for:

    <p>Identification of diseases through mutations.</p> Signup and view all the answers

    What is the primary role of dideoxynucleotides (ddNTPs) in the Sanger sequencing method?

    <p>To terminate DNA chain elongation</p> Signup and view all the answers

    During Sanger sequencing, which nucleotide types are used in greater quantities than the dideoxynucleotides?

    <p>Deoxynucleotide triphosphates</p> Signup and view all the answers

    What is the effect of capillary electrophoresis on the DNA fragments generated during Sanger sequencing?

    <p>It separates fragments according to their length</p> Signup and view all the answers

    What role does the laser play in the final step of Sanger sequencing?

    <p>It excites the fluorescent labels of nucleotides</p> Signup and view all the answers

    Why is it important that ddNTPs are present in limiting quantities during Sanger sequencing?

    <p>To ensure each fragment is terminated at different lengths</p> Signup and view all the answers

    What characteristic of shorter DNA fragments affects their movement during electrophoresis?

    <p>They migrate faster than longer fragments.</p> Signup and view all the answers

    What is the result of incorporating ddNTPs into the growing DNA chain?

    <p>The chain elongation is halted</p> Signup and view all the answers

    What causes the fluorescent peaks in the chromatogram produced during Sanger sequencing?

    <p>Light emitted from the excited ddNTP labels</p> Signup and view all the answers

    What initiates the process of bridge amplification in Illumina sequencing?

    <p>The annealing of complementary strands with flow cell oligos</p> Signup and view all the answers

    Which component is essential for the sequencing reaction in Illumina sequencing?

    <p>Fluorescently labeled dNTPs</p> Signup and view all the answers

    What is the role of the reversible terminator in the sequencer reaction?

    <p>To ensure only one type of nucleotide is added per cycle</p> Signup and view all the answers

    What occurs following the denaturation of double-stranded clonal bridges in Illumina sequencing?

    <p>Reverse strands are cleaved and removed from the clusters</p> Signup and view all the answers

    Why is next-generation sequencing (NGS) preferred over Sanger sequencing for large genomic studies?

    <p>NGS is more cost-effective and allows for broader variant detection</p> Signup and view all the answers

    What is the primary function of adapter sequences in library preparation?

    <p>To allow library hybridization to the sequencing chips</p> Signup and view all the answers

    Which statement accurately describes the amplification process in NGS?

    <p>Each library fragment generates multiple clusters on a flat surface</p> Signup and view all the answers

    What is a defining characteristic of sequencing by synthesis (SBS)?

    <p>It relies on high sequence coverage with numerous short reads</p> Signup and view all the answers

    Why does sequencing by synthesis generally exhibit a higher error rate than Sanger sequencing?

    <p>Because of incomplete removal of fluorescent signals</p> Signup and view all the answers

    What role do reversibly attached fluorescent molecules play in SBS?

    <p>They help in the optical reading of clusters</p> Signup and view all the answers

    What type of data is produced at the end of an NGS sequencing run?

    <p>Collection of DNA sequences from each cluster</p> Signup and view all the answers

    How is the final sequence determined from clusters during NGS?

    <p>Using light or fluorescence-based optical reading</p> Signup and view all the answers

    What is true regarding short-read sequencing in SBS?

    <p>It typically relies on read lengths ranging from 50 to 300 nucleotides</p> Signup and view all the answers

    What was a primary technological advancement of Next Generation Sequencing (NGS) compared to first-generation sequencing?

    <p>Utilization of high-throughput methods</p> Signup and view all the answers

    Which of the following is NOT a primary application of Next Generation Sequencing (NGS)?

    <p>Performing traditional Sanger sequencing</p> Signup and view all the answers

    Which category of NGS utilizes specific probes for identifying disease-related genetic markers?

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

    What advantage does Next Generation Sequencing (NGS) provide in terms of cost and manpower requirements?

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

    What is one of the key differences between Sequencing by Hybridization and Sequencing by Synthesis?

    <p>Synthesis involves repeated incorporation of nucleotides without ddNTPs.</p> Signup and view all the answers

    Which of the following statements about RNA sequencing (RNA-seq) is true?

    <p>It helps discover novel RNA variants and splice sites.</p> Signup and view all the answers

    Which of the following advances was crucial to the development of massively parallel sequencing technologies?

    <p>Integration of ultrafast data processing algorithms</p> Signup and view all the answers

    What is the primary benefit of sequencing cancer samples using Next Generation Sequencing (NGS)?

    <p>To study rare somatic variants and tumor subclones.</p> Signup and view all the answers

    What is the purpose of the reversible termination of nucleotides in the sequencing process?

    <p>To ensure that only one nucleotide is integrated per sequencing cycle</p> Signup and view all the answers

    In Illumina sequencing, what is the primary function of the bridge PCR amplification?

    <p>To create clonal clusters containing multiple copies of the same sequence</p> Signup and view all the answers

    What is the significance of attaching adapter sequences to DNA fragments during library preparation?

    <p>They facilitate the binding of DNA fragments to the sequencing chip</p> Signup and view all the answers

    What happens to the remaining nucleotides after one has been incorporated during the sequencing process?

    <p>They are washed away to maintain accuracy</p> Signup and view all the answers

    How does the construction of the library influence the sequencing process in Illumina technology?

    <p>It ensures uniform length and quantity of fragments</p> Signup and view all the answers

    Which of the following NGS technologies utilizes cyclic reversible termination in its sequencing process?

    <p>Illumina (Solexa) sequencing</p> Signup and view all the answers

    What is the role of the flexible linker in the bridge amplification process?

    <p>It facilitates the binding of DNA to the solid substrate</p> Signup and view all the answers

    During the Sequencing by Synthesis (SBS) process, how is the fluorescent signal interpreted?

    <p>It indicates the identity of the nucleotide incorporated at each step</p> Signup and view all the answers

    Which component is NOT involved in the cyclic reversible termination process during Illumina sequencing?

    <p>Bridge oligos</p> Signup and view all the answers

    What occurs immediately after the denaturation of double-stranded clonal bridges in Illumina sequencing?

    <p>The forward strands remain as clusters for sequencing</p> Signup and view all the answers

    Why is it important for only one of the four fluorescent dNTPs to be added per cycle in the sequencing process?

    <p>To allow accurate base calling during imaging</p> Signup and view all the answers

    Which of the following statements accurately describes bridge amplification during Illumina sequencing?

    <p>It creates double-stranded clonal bridges through nucleotide addition</p> Signup and view all the answers

    What is the main advantage of using next-generation sequencing (NGS) over Sanger sequencing in larger genomic studies?

    <p>NGS can screen more samples cost-effectively and detect multiple variants</p> Signup and view all the answers

    What is the main function of adapter sequences in the library preparation process for NGS?

    <p>They facilitate the attachment of library fragments to the sequencing surface.</p> Signup and view all the answers

    Why is a higher sequence coverage important in Sequencing by Synthesis (SBS)?

    <p>It helps in minimizing the intrinsic error rates.</p> Signup and view all the answers

    What characterizes the amplification process in NGS?

    <p>Covalently attached DNA linkers hybridize library fragments with sequencing reagents.</p> Signup and view all the answers

    What is the significance of optically reading each DNA cluster in NGS?

    <p>It facilitates the determination of sequences from multiple amplification reactions.</p> Signup and view all the answers

    What leads to the higher error rates typically observed in Sequencing by Synthesis (SBS) compared to Sanger sequencing?

    <p>Incomplete removal of the fluorescent signal, resulting in background noise.</p> Signup and view all the answers

    How does the solid surface used in NGS technology contribute to the amplification of DNA?

    <p>It provides a stable environment for covalently attaching DNA linkers.</p> Signup and view all the answers

    What is a primary outcome of having millions to billions of short DNA sequence reads in NGS?

    <p>Enhanced capability for detecting rare variants in genomes.</p> Signup and view all the answers

    What happens to library fragments after they are hybridized to the sequencing surface in NGS?

    <p>They are amplified and clustered before sequencing begins.</p> Signup and view all the answers

    What is a defining feature of Next Generation Sequencing (NGS) compared to first-generation sequencing methods?

    <p>Offers massively parallel sequencing capabilities</p> Signup and view all the answers

    Which application of NGS specifically analyses epigenetic factors?

    <p>Genome-wide DNA methylation studies</p> Signup and view all the answers

    What advancement in sequencing technology is crucial for increasing the speed and reducing costs in NGS?

    <p>Massively parallel sequencing capabilities</p> Signup and view all the answers

    Which of the following categories of NGS involves the use of specific probes for sequencing?

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

    What distinguishes sequencing by synthesis (SBS) from traditional Sanger sequencing?

    <p>It combines repeated synthesis cycles without ddNTPs</p> Signup and view all the answers

    What is one of the primary benefits of utilizing NGS for cancer sample sequencing?

    <p>Ability to study heterogeneous tumor subclones and rare variants</p> Signup and view all the answers

    What is the main limitation of sequencing by hybridization compared to sequencing by synthesis?

    <p>Dependence on specific probes affecting versatility</p> Signup and view all the answers

    What aspect of NGS has significantly improved the diversity of sequenced genomes?

    <p>Heightened efficiency in sample processing and data acquisition</p> Signup and view all the answers

    What is the result of washing away remaining nucleotides after one has been incorporated during the sequencing process?

    <p>It ensures only one nucleotide can be incorporated per cycle.</p> Signup and view all the answers

    During which step of Illumina sequencing is the library converted to single-stranded fragments?

    <p>Bridge amplification</p> Signup and view all the answers

    Which statement best describes bridge PCR in Illumina technology?

    <p>It leaves template fragments attached to the solid substrate.</p> Signup and view all the answers

    What is the overall goal of the reversible termination of nucleotides during the sequencing process?

    <p>To allow for precise identification of each nucleotide incorporated.</p> Signup and view all the answers

    What is the purpose of denaturing DNA fragments in the library preparation step of Illumina sequencing?

    <p>To facilitate the binding of adapters to DNA.</p> Signup and view all the answers

    Which technology utilizes cyclic reversible termination during sequencing?

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

    In the context of sequencing by synthesis, what does the term 'clonal cluster' refer to?

    <p>A region where multiple copies of the same template DNA are amplified.</p> Signup and view all the answers

    Which of the following describes a primary challenge in sequencing by synthesis relative to Sanger sequencing?

    <p>Inherent higher error rates.</p> Signup and view all the answers

    Study Notes

    DNA Sequencing

    • The process of determining the order of bases in a length of DNA.
    • It has revolutionized our understanding of Genetics.
    • Key applications are:
      • Tackling Human Disease
      • Identifying variations and polymorphisms in the human genome
      • Studying evolution

    Sanger Sequencing

    • Also known as "chain termination method".
    • Classified as a First Generation Sequencing method.
    • Developed in 1977 by Fred Sanger and his colleagues - the first to sequence a whole genome.
    • The genome of the virus phiX174, which infects bacteria, was just over 5000 bases.

    Sanger Sequencing Method

    • Utilizes high fidelity DNA-dependent polymerase.
    • Creates a complementary copy to a single stranded DNA template.
    • Employs a single primer, complementary to the template, which initiates DNA synthesis from the 3' end.
    • Uses dideoxy nucleotides (ddNTPs) which resemble DNA monomers but lack a 3' hydroxyl group, causing chain termination after incorporation.
    • Each ddNTP is fluorescently labeled, allowing for automatic detection.

    Principle of Sanger Sequencing

    • A mixture of dNTPs (normal nucleotides) and ddNTPs (dideoxy nucleotides) is used.
    • The ddNTPs are present in limiting amounts, which ensures that the chain termination occurs randomly at any point.
    • The DNA fragments containing the ddNTPs are separated based on size by gel electrophoresis.
    • Each ddNTP is labelled with a specific fluorescent dye so that the sequence can be read, as the fragments pass through a laser beam.

    Steps in Sanger Sequencing

      1. Amplification of DNA by PCR: The target DNA is amplified using PCR to create multiple copies.
        • The double helix is then denatured using heat separating the two strands, which serve as templates for DNA synthesis.
      1. Generation of DNA fragments of varying lengths by chain termination:
        • The DNA template is combined with a primer, DNA polymerase, dNTPs, and a small amount of labeled ddNTPs.
        • Each ddNTP is tagged with a distinct fluorescent dye.
        • The process generates DNA fragments of varying lengths, each ending with a labeled ddNTP.
        • No additional nucleotides can be added to the chain after a ddNTP is incorporated, leading to termination.
      1. Capillary electrophoresis:
        • The fragments are separated by capillary electrophoresis based on size (shorter fragments move faster), creating a sequence of DNA fragments tagged with fluorescent dyes.
      1. Data analysis:
        • The fluorescently labeled ddNTPs are irradiated with a laser beam.
        • The excited dye emits light, which is detected.
        • The sequence of colored peaks in the electropherogram corresponds to the sequence of nucleotides in the target DNA.

    Human Genome Project

    • Human Genome Project was the first major foray into DNA sequencing
    • Used first generation sequencing, Sanger Sequencing (Chain Termination method)
    • Took 13 years to complete
    • Cost $3 billion
    • Completed in 2003

    Next Generation Sequencing (NGS)

    • NGS implies the next step in the development of DNA sequencing technology
    • Introduced in 2004 and 2006
    • Second Generation Sequencing is a term associated with NGS

    Next Generation Sequencing (NGS)

    • NGS is massively parallel sequencing technology
    • Offers ultra-high throughput, scalability, and speed.
    • Used to determine the order of nucleotides in:
      • entire genomes
      • Targeted regions of DNA or RNA
    • NGS has revolutionized biological sciences, allowing for wide variety of applications

    Next Generation Sequencing (NGS)

    • Biggest advances in genome sequencing are due to increased speed and accuracy
    • NGS decreases cost and manpower requirements
    • NGS decreased cost per megabase
    • Increased the number and diversity of sequenced genomes

    Applications of NGS

    • 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
      • Genome-wide DNA methylation
      • DNA-protein interactions
    • Sequence cancer samples to study rare somatic variants, tumor subclones, etc.
    • Identify novel pathogens

    Categories of NGS

    • Two major categories of NGS:
      • Sequencing by hybridization
      • Sequencing by Synthesis (SBS)

    Sequencing by hybridization

    • Uses specific probes to interrogate sequences
    • Used in diagnostic for:
      • Identifying disease-related SNPs
      • Identifying gross chromosome abnormalities – rearrangements, deletions, duplications, copy number variants (CNVs)

    Sequencing by Synthesis (SBS)

    • Further development of Sanger sequencing
    • SBS does not use ddNTPs
    • Uses a combination of repeated synthesis cycles, and methods to incorporate nucleotides into a growing chain

    Similarities between different NGS technologies

    • Sample preparation:
      • Requires library obtained by amplification or ligation with custom adapter sequences
      • Adapter sequences allow library hybridization to sequencing chips
      • Adapters provide a universal priming sire for sequencing primers

    Similarities between different NGS technologies

    • Sequencing machines:
      • Each library fragment is amplified on a solid surface – either beads or flat silicon derived surface
      • This is done using covalently attached DNA linkers that hybridize library adapters
      • Amplification creates clusters of DNA – each originating from a single library fragment
      • Each cluster acts as an individual sequencing reaction
      • The sequence from each cluster is optically read – either by light or fluorescence
      • Each machine has its own cycling condition

    Similarities between different NGS technologies

    • Data output:
      • Each machine provides raw data at the end of sequencing run
      • Raw data = collection of DNA sequences generated at each cluster
      • The data is further analyzed to provide meaningful results

    Sequencing by Synthesis (SBS) properties

    • Relies on shorter reads (300-500 bp)
    • Generally has intrinsically higher error rate relative to Sanger
      • Due to incomplete removal of fluorescent signal which can cause higher background noise levels
    • Relies on high sequence coverage – “massively parallel sequencing”
      • Of millions to billions of short DNA sequence reads (50 – 300 nucleotides )
      • = Short Read Sequencing

    Sequencing by Synthesis (SBS) properties

    • 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 - ensures only one nucleotide is incorporated per cycle

    Sequencing by Synthesis (SBS) Process

    • All four nucleotides are added to the sequencing chip
    • Single nucleotide is incorporated into the sequence
    • Remaining nucleotides are washed away
    • Fluorescent signal is read at each cluster and recorded
    • Both Fluorescent molecule and terminator group are cleaved and washed away
    • Process is repeated 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)

    Illumina Sequencing – Step 1: Library preparation

    • First Step: break up DNA into more manageable fragments (~200 – 600bp)
    • Short sequences of DNA – adapters – are attached to DNA fragments
    • DNA fragments attached to adapters are denatured (made single stranded)
    • Libraries are constructed to give 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 that needs to be sequenced
    • An in vitro constructed adapter flanked library is PCR amplified
    • Both primers densely coat the surface of the solid substate – attached at their 5’ end by a flexible linker
    • Amplification products from the template library remain locally attached near the point of origin

    Illumina Sequencing – Step 2: Bridge amplification

    • At the end of the PCR – each clonal cluster contains ~1000 copies of a single member of the template library
    • DNA fragments attached to adapters are then made single-stranded
    • Once prepared – DNA fragments are washed across the flow cell (also known as the sequencing chip)
    • Complementary DNA binds to primers on the surface of the flow cell
    • DNA that doesn’t attach is washed away

    Illumina Sequencing – Step 2: Bridge amplification

    • Complementary strand of DNA fragment in library is synthesized
    • Complementary strand folds over and anneals with the other type of flow cell oligo – forms a bridge
    • Double stranded bridge is denatured – forming two single strands attached to the flow cell
    • Process of bridge amplification repeats
    • More clones of double stranded bridges formed

    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

    • Bridge amplification – Clonal clustering
      • Components:
        • Primers
        • dNTPs
          • Labelled with a fluorescent dye
          • Contains a reversible terminator (Trinitrogen = N3)
        • DNA polymerase

    Illumina Sequencing – Step 3: Sequencing Reaction

    • Bridge amplification – Clonal clustering
      • Process:
        • Cyclic reversible termination
        • Only one of four fluorescent dNTPs added per cycles
        • Images of clusters are captured after incorporation of each nucleotide
        • After imaging, fluorescent dye and terminator are cleaved and released

    Illumina Sequencing – Step 3

    • Trinitrogen (N3)
    • Fluorescent dye

    Illumina Sequencing – Step 3

    • Image captured
    • Fluorescent dye and terminator are cleaved and released

    When to use NGS vs Sanger Sequencing

    • Sanger sequencing:

      • Good choice when investigating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer)

    •  NGS

      • Allows you to screen more samples cost-effectively
      • Allows detection of multiple variants across targeted areas of the genome
      • Used when Sanger sequencing approaches would be too costly and time-consuming

    Human Genome Project

    • The Human Genome Project was the first major project to sequence DNA.
    • It used first-generation sequencing, specifically Sanger Sequencing.
    • Took 13 years to complete.
    • Cost $3 billion dollars to complete.
    • Completed in 2003.

    Next Generation Sequencing (NGS)

    • Implies the next step in the development of DNA sequencing technology.
    • Second-Generation Sequencing was introduced in 2004 and 2006.
    • Provides high-throughput sequencing.

    Next Generation Sequencing (NGS)

    • Massively parallel sequencing technology offers ultra-high throughput, scalability, and speed.
    • Used to determine the order of nucleotides in:
      • Entire genomes
      • Specific regions of DNA or RNA
    • Revolutionized biological sciences, leading to a wide variety of applications.

    Next Generation Sequencing (NGS)

    • The biggest advances in genome sequencing are due to increased speed and accuracy.
    • This results in decreased cost and manpower requirements.
    • NGS has decreased the cost per megabase.
    • Increased the number and diversity of sequenced genomes.

    Applications of NGS

    • 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:
      • Genome-wide DNA methylation
      • DNA-protein interactions
    • Sequence cancer samples:
      • Study rare somatic variants
      • Analyze tumor subclones
    • Identify novel pathogens.

    Categories of NGS

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

    Similarities between different NGS technologies

    • Sample Preparation:
      • Requires a library obtained by:
        • Amplification or ligation with custom adapter sequences.
        • Adapter sequences allow library hybridization to the sequencing chips.
        • Provide a universal priming site for sequencing primers.
    • Sequencing machines:
      • Each library fragment is amplified on a solid surface - either beads or flat silicon derived surface.
      • This is done using covalently attached DNA linkers that hybridize library adapters.
      • Amplification creates clusters of DNA - each originating from a single library fragment.
        • Each cluster acts as an individual sequencing reaction.
        • The sequence from each cluster is optically read - either by light or fluorescence.
        • Each machine has its own cycling conditions.
    • Data output:
      • Each machine provides raw data at the end of the sequencing run.
      • Raw data = collection of DNA sequences generated at each cluster.
      • This data is further analyzed to give meaningful results.

    Sequencing by Synthesis (SBS) properties

    • Relies on shorter reads (300-500 bp).
    • Generally has a higher error rate than Sanger.
      • Due to incomplete removal of fluorescent signal which can cause higher background noise levels.
    • Relies on high sequence coverage - "massively parallel sequencing".
      • Millions to billions of short DNA sequence reads (50 - 300 nucleotides).
        • Short read sequencing.

    Sequencing by Synthesis (SBS) properties

    • Utilizes step-by-step incorporation of reversibly fluorescent and terminated nucleotides.
      • 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 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.
    • The remaining nucleotides are then 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 is repeated 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)

    • Cyclic reversible termination is a method of sequencing.

    Illumina Sequencing – Step 1: Library Preparation

    • The first step is to break up the DNA into more manageable fragments (~200 – 600bp).
    • Short sequences of DNA, called adapters, are then attached to the DNA fragments.
    • The DNA fragments attached to adapters are denatured (made single stranded).
    • Libraries are constructed to give 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 that needs to be sequenced.
    • 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.
      • Because of this, amplification products from the template library will remain locally attached near the point of origin.

    Illumina Sequencing – Step 2: Bridge amplification

    • At the end of the PCR, each clonal cluster contains ~1000 copies of a single member of the template library.
    • DNA fragments attached to adapters are then made single-stranded.
    • Once prepared, DNA fragments are washed across the flow cell (also known as the sequencing chip).
    • Complementary DNA binds to primers on the surface of the flow cell.
    • DNA that does not attach is washed away.

    Illumina Sequencing – Step 2: Bridge amplification

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

    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

    • Contains the following components:
      • Primers
      • dNTPs:
        • Labelled with a fluorescent dye.
        • Contains a reversible terminator (Trinitrogen = N3).
        • Contains a DNA polymerase.

    Illumina Sequencing – Step 3: Sequencing Reaction

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

    Illumina Sequencing – Step 3

    • An overview of the cycle, including the role of Trinitrogen.

    Illumina Sequencing – Step 3

    • An overview of the cycle.

    Illumina Sequencing: Overview

    • A comprehensive illustration of the entire sequencing process.

    When to use NGS vs Sanger Sequencing

    • Sanger sequencing:
      • Good choice when investigating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer).
    • NGS:
      • Allows you to screen more samples cost-effectively.
      • Allows detection of multiple variants across targeted areas of the genome.
      • Used when Sanger sequencing approaches would be too costly and time-consuming.

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    Related Documents

    DNA Sequencing Lecture Notes PDF
    DNA Sequencing: Next Generation Sequencing (NGS) Lecture PDF

    Description

    Explore the fascinating world of DNA sequencing, including the revolutionary Sanger sequencing method. Understand its applications in genetics, human disease, and evolution. Test your knowledge on the technical aspects and historical significance of this groundbreaking technique.

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