DNA Sequencing Techniques

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.
• 2. 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.
• 3. 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.
• 4. 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.