NGS Techniques

• NGS


  Any method of DNA sequencing that has replaced the Sanger method
• Features of NGS

  • Thousands to millions of DNA molecules can be sequenced simultaneously (in parallel)
  • Quicker: human genome can be sequenced in under 1 hour
  • Cheaper: human genome can be sequenced for under £1,000
  • Accurate: comparable to Sanger > at around 99.9%
• The HGP cost $3bn & took 13yrs
• Sanger method


  Requires size determination, constraining the number of DNA clones that could be sequenced at one time
• 2 KEY FEATURES OF NGS

  • Clonal amplification of DNA fragments using massively parallel PCR-based techniques
  • Sequencing of the DNA clones, in parallel, using a light detection method
• Pyrosequencing set-up


  1. DNA is broken up into fragments of ~100bp

  2. Each fragment is denatured to form ssDNA

  3. Adaptors (known DNA sequence) are added to the ends of each fragment (for primer annealing)

  4. Each ssDNA fragment is attached to a microscopic bead

  5. PCRs proceed on each bead

  6. Each individual droplet PCR reaction bead is spread onto pico-titer plates

  7. Perform pyrosequencing in wells, using primers complementary to the adaptors
• Pyrosequencing sequencing


  1. A single DNA strand is synthesised 1 base at a time

  2. If an activated nucleotide is added, light is emmitted and detected to determine the complementary base sequence

  3. Each activated nucleoide is iteratively added until a flash is detected

  4. Pyrophosphate is released (PPi)

  5. In the presence of APS, the enzyme ATP sulfurylase converts PPi to ATP

  6. Luciferase combines ATP and Luciferin into Oxyluciferin + Light

  7. Apyrase: added after each nucleotide addition to degrade it
• Pyrosequencing key difference?


  Uses activated nucleotides, with additional phosphates
• Pyrosequencing advantage 


  Cheaper and quicker than Sanger, when applied to genome-scale projects
• Pyrosequencing limitations


  • Not as accurate as Sanger. Higher error-rate
  • Not well-suited for larger genome sequencing
  • Only short stretches of DNA can be analysed
• Illumina sequencing


  Most commonly used sequencing method. Accurate and cheap
• Illumina sequencing


  1. Adaptors, complementary to the primers, are attached to both ends of the DNA fragments

  2. PCR primers: billions are attached to the surface of the glass slide (known as a flow cell)

  3. The adaptor motif of the DNA fragment binds to complementary primers in a lane of the flow cell

  4. Polymerase creates a complimentary template of the hybridised fragment

  5. The newly synthesised double stranded molecule is denatured and the original template (the fragment that bound to the primer) is washed away. The clone remains bound

  6. Clonal amplification: strand folds and hybridises to the second type of primer on the flow cell

  7. Polymerases synthesise a complementary strand, to form a double stranded bridge

  8. The bridge is denatured, resulting in 2 single stranded copies of the DNA fragment, tethered to the flow cell. Process repeats and occurs simultaneously for millions of clusters
• Illumina sequencing


  1. Modified nucleotides with a chemically-cleavable fluorescent dye attached and a hydroxyl group that contains a reversibly bound terminator group to block the addition of the next nucleotide, until chemically reversed into a 'normal' hydroxyl group – giving off fluorescence when cleaved

  2. Sequence-by-synthesis (SBS): nucleotides compete for addition to the growing chain. They're added 1-by-1

  3. The reaction terminates after the correct nucleotide is incorporated

  4. Upon incorporation, the flurophore is excited and emits a fluorescent signal

  5. Chemical is added to reverse the terminator group back to a normal hydroxyl group to allow for more nucleotides to be added

  6. Reads are limited to ~200nt
• Illumina sequencing advantages


  • Higher throughput (more sequence yielded per unit time and cost)
  • More accurate than other latest NGS
• Latest NGS techniques


  • Allow DNA to be sequenced without PCR amplification
  • Generate much longer sequence reads
  • Sequence data is obtained in real-time
• SMRT Sequencing


  1. Zero-mode waveguide (ZMW): a nanophotonic confinement structure that guides light energy into a volume smaller than the wavelength of light – zeptoliter volumes

  2. Inside each ZMW is a single DNA polymerase, immobilised at the bottom of the well

  3. Light penetrates and encompasses the DNA polymerase to allow for monitoring of light flashes

  4. Phospho-linked fluorescent nucleotides: each nucleotide has a corresponding fluorescent dye molecule, which is attached to the phosphate chain

  5. Nucleotides are in the surrounding solution. Not in the ZMW long enough to be excited

  6. When a nucleotide is incorporated, the fluorescent dye is held in the ZMW long enough to be excited, giving off a colour. It's subsequently cleaved off and its fluorescence is no longer observable
• SMRT Sequencing advantages


  Increase read length into the kilobases. No clone of template needed
• SMRT Sequencing features


  Reduced throughput and accuracy. More expensive. Illumina is more often used
• Nanopore


  1. Relies on ion channels found in cell membranes (often bacterial)

  2. Ion channel is embedded into an inert membrane and bathed in an electrolyte solution that has a potential difference applied

  3. Flow of ions through the pore generates a steady current

  4. As a nucleotide enters through the pore, the current is altered slightly. Each nucleotide alters the current flow in a different way, producing a signature deflection

  5. The change in current is detected

  6. Motor enzyme interacts with the ion channel and feeds DNA through the pore
• Nanopore


  • Higher throughput than SMRT
  • More accurate than SMRT
  • Still, currently not as efficient as Illumina
• Parallel sequencing = high throughput approaches that can process many sequencing reactions simultaneously
• What is parallel sequencing?


  High throughput method to sequencing that allows many reactions to occur simultaneously
• Size determination = determining the length (size) of DNA fragments using electrophoresis
• What is an oligonucleotide?


  Short sequence of nucleotides (usually between 13-25)
• What is the purpose of an oligonucleotide?

  Hybridise to complementary target DNA/RNA. Used in PCR, DNA sequencing, etc.
• PCR primers are short pieces of single stranded DNA used to initiate replication of a specific region of DNA by serving as a start site for DNA polymerase
• Probes are short pieces of single stranded DNA or RNA used to detect presence of particular sequences within larger molecules
• DNA probes can be labelled with radioactive tags, fluorophores, biotin, enzyme labels, etc.
• Fluorescently labeled probes bind to their complement target DNA/RNA and emit light when the fluorophore is excited by light.
• How does a fluorophore probe emit light?


  Photon excites the flurophore and its electrons are energised.
• SMRT Preparation

  • Sample is isolated
  • Adaptors are ligated either end to form a circular molecule
  • Primer + polymerase added to the template
  • Each well (ZMW) houses a single molecule of DNA + Polymerase.
  • There are millions of ZMWs, allowing for simultaneous sequencing of DNA