week 8 Flashcards
(12 cards)
describe sanger sequencing
- has been the major method for last 45 years
- revolutionised molecular biology (human genome draft)
- main change is number of samples sequenced has increased
- however length of reads per run has only improved slightly over the years
described sanger sequencing limitations
- Maximum read length usually <1000bp
- High cost per base
- Long experimental set up times
- High DNA concentrations needed
- Some regions are unsequenceable (GC rich, etc)
describe sequencing by synthesis
- Single DNA molecules replicated
- Replicated DNA made single stranded
- Replicates (clonal amplifications) are ‘fixed’ to a specific location
- Sequencing occurs using a DNA polymerase based method (the sequence is analised as sequencing happens!)
preparation of a library for sequencing
The first step is to:
- break up target DNA into small fragments (Shotgun: sonication, enzymatic; 50-300bp)
- attach adaptors to the end of the sequence (made like ‘primers’ but they are double stranded)
- and then make fragments single stranded
describe single stranded DNA fragments are bound to flow cell
- Single stranded fragments are attached to surface of a ‘flow cell’(with 8 lanes)
- Flow cell can support 8 separate samples
- On flow cell surface are millions of small sequences (primers) that are complementary to both adaptors
describe how single stranded DNA fragments are bound to target adapters on flow cell
- left with lots of target DNA fragments scattered across plate with one free adaptor end
- free end of fragment with adaptor binds with complementary surface ‘primers’
- this forms a physical bridge
describe the formation of double stranded molecule
- add unlabelled nucleotides and ‘PCR’ reagents
- complementary strand is built to create a double stranded bridge
- only one end of each strand is actually bound to surface
describe denaturation and repeat to make clusters
- break the DNA bridge by simply denaturing it
- left with both strands now separated on the flow cell, bound at one end again
- repeating this bridge building-breaking process creates clusters of clonally amplified DNA templates across the flow cell surface
- each cluster contains half of one strand type and half the complementary strand type
describe the removal molecules attached by one type of adaptor
- remove those strands attached to flow cell by one type of adaptor
- left with DNA fragment clusters that are only attached by one adaptor type
- these act as sequencing templates
- add sequencing primer that complements all free ends of DNA fragments
describe the addition of reversible terminators and measurement of fluorescence
- reversible terminators are chemically modified nucleotides
- once incorporated onto a template, no further nucleotides can be added until the chemical modification is removed
- similar in principle to dideoxynucleotide triphosphates used in Sanger sequencing except effect is reversible
- the ones used in illumina sequencing also releases a fluorescent signal specific to the nucleotide when excited by a laser
- all four reversible terminator types are added simultaneously to the flow cell
- a complementary nucleotide is added to the first free nucleotides of the templates present in each cluster
- the nucleotide added will depend on the sequence of each specific cluster
- the flow cell then undergoes laser excitation
- the laser excitation caused each cluster to fluoresce based on the specific nucleotide that was added
- a picture of the flow is taken to capture the fluorescent patterns
- fluorescent spots on image signify each cluster
- the colour of the fluorescent specifies the nucleotide that was added in each cluster
- remove terminators (removes fluorescence)
- process from first round of sequencing is repeated many times to sequence each fragment
- before each iteration, the chemical modification is removed from the reversible terminators from the previous round of sequencing
- each round of sequencing produces a separate flow cell image
- all flow cell images will have fluorescent spots at the exact same positions
- images will differ by the pattern of colours
- by analysing each position separately from image to image you can build up the entire sequence of each fragment
- we will end up with lots of short random sequences which can be assembled by comparison to a reference sequence
- sequence fragments are between 50-300bp
- up to 10 billion fragment reads per run is possible
describe the advantages and disadvantages of illumina
advantages:
- no problem with homopolymer regions as it is base position by base position sequencing
- simultaneous base addition reduces mis-incorporation rate
- cheaper per base than other methods
- can sequence >600GB per run
- requires less initial start DNA than some other approaches
disadvantages:
- error rate is still greater than Sanger sequencing
- equipment can be very expensive
- sometimes can require high concentrations of DNA
describe NGS for better cancer therapeutics
- cancer is one end-product of somatic evolution, in which a single clonal lineage acquires a complement of driver mutations that enables the cells to evade normal constraints on cell proliferation, invade tissues and spread to other organs
- somatic cells accumulate mutations throughout life
- mutations can be classified as:
- confer a selective advantage on the cell, increasing survival or proliferation
- those that are selectively neutral
- those that are disadvantageous to the cell and result in its death or senescence
