Next Generation Sequencing Part One

Question 1

Outline NGS.

Answer 1

• It allows millions of sequencing reactions to be carried out in parallel.
• Sequencing is of clonally amplified DNA templates or single DNA molecules.
• Disadvantage: read length is shorter than sanger and the error rates for individual NGS reads is greater.
• Accuracy in NGS is achieved by sequencing a given region multiple times e.g. 40 times (reads) and a consensus sequence is generated.
• Hence the initial advantage is off set by needing to sequence the same region so many times but despite this cost savings can still be made.
• To assemble, align, and analyse NGS data requires an adequate number of overlapping reads.

Question 2

What are the main NGS sequencing chemistries?

Answer 2

3 main types:

• Sequencing by synthesis
• Sequencing by ligation
• Other developing techniques e.g. nanopore

Question 3

What are the common library preparation methods for NGS?

Answer 3

1) . Fragmentation
- Sonication
- Nebulization
- Shearing

2) . End-repair
- Size selection
- Blunt-end or A-overhang

3) . Adaptor Ligation
- Ligation
- Purification

Question 4

Outline library preparation methods for Illumina.

Answer 4

• The starting material for library preparation can be long range PCR products or any other region of interest.
• In the process of library preparation the DNA is further amplified.
• For Illumina the clonal amplification steps take place in situ on the surface of the flow cell itself rather than in a separate emulsion PCR reaction.
• Similar to other platforms the DNA library is first ligated oligonucleotide adapters which incorporate a sequence complimentary to anchor oligonucleotides which are covalently linked to the surface of the flowcell.
• After annealing to the anchor DNA nucleotides the template DNA molecules are clonally amplified in a modified isothermal PCR reaction termed bridge PCR. in which the DNA molecules are free to flex and form a bridge with an adjacent anchor oligonucleotide.
• The process results in the generation of more than 50 million individual clusters containing over 1000 copies of clonally amplified molecules on the surface of the flow cell.
• Next the clusters are denatured to provide a single stranded template. Sequencing primer oligonucleotide is hybridised to the DNA.
• During each sequencing reaction the clonally amplified clusters are exposed to DNA polymerase and a mixture of 4 nucleotides each labelled with a unique fluorescent label.

Question 5

Outline library preparation methods for Roche FLX 454.

Answer 5

• DNA fragmented and then adapters are joined at either end of the strands.
• The fragmented DNA is combined with beads in the presence PCR reagents such that there is one molecule per bead.
• Emulsification takes place in water oil mixture to provide physical separation of the components into individual microreactors.
  (Emulsification PCR (emPCR) in water-oil mixture).
• After amplification the emulsion is broken with a solvent and the beads are enriched by incubation with streptavidin-coated magnetic beads to selectively purify beads containing a biotin label amplified product.
• A sequencing primer is annealed to the DNA bound to the beads and the beads are loaded onto a fibre optic titre plate containing millions of individual wells - 1 well = 1 bead.
• Emulsion PCR is also carried out for the solid system.

Question 6

Outline the fluorescent reversible dye terminator system used in illumina NGS.

Answer 6

• During the sequencing cycle a single fluorescent labelled dNTP is added to the nucleic acid chain.
• The nucleotide label serves as a terminator of polymerisation so after each dNTP incorporation the fluorescent dye is imaged to identify the base and then enzymatically cleaved to allow incorporation of the single nucleotide.
• Since all 4 reversible terminators bound to dNTPs are present as single separate molecules natural competition minimises incorporation bias.
• Base calls are made directly from single intensity measurements during each sequencing cycle which greatly reduces mismatch rates compared to other technologies.
• The end result is highly accurate base by base sequencing that greatly reduces sequencing context specific errors enabling robust base calling across the genome including repetitive regions and within polymers.

Question 7

Outline the Roche FLX pyrosequencing reaction.

Answer 7

• A sequencing primer is annealed to the DNA bound to the beads and the beads are loaded onto fibre optic picotitre plates containing millions of individual wells.
• To ensure 1 sequencing read per well of the plate each has approximately the diameter of a single bead.
• Pyrosequencing takes chemical advantage of the pyrophosphate molecule liberated by the addition of a dNTP during the extension step.
• The pyrophosphate molecule is converted to ATP through the action of Sulfurylase - the ATP molecule is subsequently used by Luciferase to convert luciferin to oxyluciferin. This reaction generates light which can be measured and quantified by a highly sensitive camera within the instruments.
• For short single nucleotide repeat stretches the intensity of the light emitted is proportional to the number of oligonucleotides incorporated. However, for longer homo-polymer stretches >8nts the signal begins to show loss of linearity with a concomitant rise in base call errors.
• The fluorescence is proportional to the number of nucleotides incorporated - this works up until about 6 or 8 nucleotides (e.g. AAAAAAAA) but after this a detection of homopolymer tracts can be a problem with this technique.

Question 8

Outline Ion Torrent.

Answer 8

• Ion torrent works by measuring the voltage changes induced by the release of hydrogen ions when a base is incorporated.
• The Ion Torrent does not require the use of fluorescent nucleotides which can be expensive.
• Sequencing by synthesis occurs by sequential addition of unmodified nucleotides.
• Unlike pyrosequencing, sequencing occurs by direct detection (hydrogen ions are released as a result of the synthesis reaction) - translated into voltage signal and subsequently into a base call.
• No cameras, light sources, or scanners involved - only a digital voltage change is used.

Question 9

Outline the SOLiD NGS method.

Answer 9

• The SOLiD technique differs from other NGS platforms in that the sequencing is synthetically determined by a probe ligation method. It does not utilise a DNAP to incorporate nucleotides.
• Similar to the 454 approach the first step is an emulsion PCR to generate a clonally amplified adapter modified DNA molecule bound to a bead.
• Relies on 16 8-mer oligonucleotide probes each with one of 4 fluorescent dyes attached at the 5’ end.
• Each octomer consists of 2 probe-specific bases and 6 degenerate bases.
• The sequencing reaction commences by binding of the primer to the adapter sequence and then hybridisation of the appropriate probe. This hybridisation is guided by the 2 probe specific bases and upon annealing is ligated to the DNA primer sequence through a DNA ligase.
• Unbound oligonucleotides are washed away then the signal is detected and recorded. After that, the fluorescence signal along with the last 3 bases of the octomer probe are cleaved and then the next cycle commences.
• After approximately 7 cycles of ligation the DNA strand is denatured and another sequencing primer offset by 1 base to the previous primer is used to repeat these steps. In total, 5 sequencing primers are used.
• The major disadvantage of this technology is the very short sequencing reads generated.

Question 10

Watch bookmarked NGS tech overview.

Answer 10

Bookmarked youtube video.