types of sequencing Flashcards
1
Q
what is next generation sequencing
A
- massively parallel sequencing
- new technique which speeds up process
- more automated
2
Q
where does next generation sequencing take place?
A
plastic slide called flow cell
3
Q
how does next generation sequencing work?
A
- millions of fragments attached to cell are replicated on slide by PCR to form clusters of identical fragments
- still uses coloured terminator bases
4
Q
how is next generation sequencing different to original sequencing method?
A
- faster
- reads clusters of fragments at once rather than individually
5
Q
what is pyrosequencing?
A
- fragments added to a bead and amplified by PCR
- add bead to well with enzymes called sulfurylase and luciferase
- add a nucleotide
- if A complementary base pairs with fragment DNA, enzymes convert energy into a flash of light
- wash well
- add second nucleotide
(nucleotides have to be added separately)
6
Q
what happens if there is no base pairing in pyrosequencing?
A
no light
7
Q
what happens if there is 2 consecutive pairs in pyrosequencing?
A
twice the light
8
Q
what is nanopore sequencing?
A
- uses flow cells which contain tiny holes called nanopores
- each nanopore corresponds to its own electrode connected to a channel and sensor chip
- this measures the electric current that flows through nanopore
- when a molecule passes through, the current is disrupted to produce a characteristic squiggle
- squiggle is decoded using basecalling algorithms to determine DNA or RNA sequence in real time
