18.02.14 NGS technologies Flashcards
What are the different types of enrichment methods?
PCR-based methods
Hybridisation methods
Give examples of highly-multiplexed PCR enrichment kits
- Ampliseq (Life Technologies)
- GeneRead DNA seq targeted panels (Qiagen)
- Truseq/Nextera (Illumina)
What are the disadvantages to using amplicon selection assays?
Inaccurate copy number calculation as only unique reads should be used and it is difficult to separate out the PCR duplicates.
Give examples of target enrichment methods that use hybridisation.
Solution based capture of target regions.
Agilent SureSelect
Haloplex
TruSight
Roche EZ-Cap
What are the stages of hybridisation based capture methods?
- Fragment gDNA (shearing)
- Prepare library: Tag framents with sequence-ready adaptors and barcodes
- Capture libraries using RNA or DNA based oligonucleotides
- Oligos anneal to specific regions of the genome to result in a tiling of captured fragments representative of the entire ROI
- Clean up of capture DNA is aided by use of oligo bairs conjugated to biotin which can be bound by paramagnetic beads coupled to streptavidin.
What are the stages in the Haloplex system?
- Restriction digestion of gDNA (8 double enzyme reactions) generating non-random fragments avoids the need for a library prep stage.
- Ends of fragments are captured by the annealing of partially double-stranded biotinylated oligonucletodies.
- Bound molecules captured using steptavidin coated magnetic beads.
- Central portion of the probe contains PCR primer binding sites, appropriate barcodes and sequencing adaptors
- Ligation results in the formation of a closed circle
- Subsequent PCR using a common pair of primers generates a linear library of enriched fragments with appropriate sequencing adaptors at the ends.
- Due to opposing orientation of the primers used only circular molecules will be amplified enhancing specificity.
What factors will influence the choice of enrichment method?
- Cost
- Equipment
- Panel size
- TAT
- Sample type
What is the difference between 2nd and 3rd generation sequencing platforms?
Second generation platforms utilise an amplification step prior to sequencing the library molecules, unlike the single molecule sequencing performed by third generation platforms.
Give examples of second generation sequencing platforms.
Ion Torrent (Life technologies)
MiSeq/HiSeq/NextSeq (illumina)
454 (Roche)
SOLiD (Life Technologies)
What are the common features of second generation sequencing platform?
1) A series of automatically coordinated repeating chemical reactions typically carried out in a flow cell or compartment which house the immobilised templates and necessary reagnets.
2) Most platforms use sequencing by synthesis - a repeated cyclical process which occurs within the flow cell and consists of nucleotide addition, washing and signal detection.
Describe the stages in Illumina HiSeq etc sequencing.
- Library prep: fragmentation of high molecular weight DNA, enzymatic trimming and adenylation of fragment ends and ligation of specific adaptors.
- Pricisely diluted solution of library fragments is amplified in situ of the flow cell surface by a bridge amplification step to produce clusters for sequencing.
- Fragments ends carrying the same adaptor are released, which is then primed with a complementary synthetic DNA primer to provide free 3’OH groups that can be extended in subsequent sequencing reactions.
- In reversible dye-terminator sequencing, all four nucleotides are provided in each cycle because each nucleotide carries an identifying fluorescent label.
- Sequencing occurs as single-nucleotide addition reactions because a blocking groups exists at the 3’OH position of the ribose sugar, preventing additional base incorporation reactions by the polymerase.
Each step = Nucleotide added by polymerase, unincorporated nucleotides washed away, flow cell is imaged on both inner surfaces to identify each cluster that is reporting a fluorescent signal, the fluorescent groups are chemically cleaved and the 3’OH is chemically deblocked.
Describe the structure of the Illumina microfluidic conduit.
Flow cell composed of flat glass with eight microfluidic channels, each decorate by covalent attachment of adaptor sequences complementary to the library adaptors.
What are the principles of Ion Torrent sequencing.
Uses the release of H+ as a by-product of the incorporation of a nucleotide into a strand of DNA by a polymerase - measurable change in the potential difference.
Uses a chip with a high-density array of micro-machined wells, each holding a DNA template.
Beneath the well is an ion-sensitive layer and beneath that a proprietary ion sensor.
Describe the stages in Ion Torrent.
- Library molecules are first clonally amplified onto IonSphere particles by emulsion PCR
- Template-bearing beads are enriched through a magnetic bead based process
- Sequencing primers and DNA polymerase are bound to the templates and pipetted into the chip’s loading port
- Individual beads are loaded into individual sensor weels by centrifugation
- 4 non-fluorescent nucleotides are added individually in a sequential order - incorportation of a nt into nascent strand increases the length of the seq primer by one base.
- Incorporation = hydrolysis of nucleotide triphosphate and net production of a H+ ion in that flow.
- This shifts the pH in surrounding solution proprotional to the number of nt added (0.02pH units per base) which is detected by the semiconductor sensor and converted to a voltage and digitised by off-cip electronics.
- Wash step after flow of each nt. Small size of wells allows diffusion into and out of well in 1/10 of a seconds and eliminates the need for enzymatic removal of reagents.
What are the advantages and disadvantages of Illumina sequencing technologies (MiSeq etc)?
A: Most widely used platform.
Performs well at homopolymer regions
D: Low multiplexing capabilities of samples. Sequencer more expensive than IonTorrent.
