Lecture 16 - High-Throughput Sequencing Flashcards
how does Sanger sequencing work
modification of PCR, fluorescently labelled dideoxy nucleotides are added in
what are dideoxy nucleotides
chain terminating nucleotides
when is Sanger sequencing used
sequencing individual genes, not genomes
what type of sequencing is Illumina
short read
what type of sequencing is PacBio and Oxford nanopore
long read
what are DNA fragments ligated to in Illumina sequencing
adapters, which bind to primers fixed to a substrate
what follows low-density binding to substrate for Illumina sequencing
bridge PCR amplification
how is bridge PCR amplification done in Illumina sequencing
adding unlabelled nucleotides and DNA polymerase to the sample
how is single stranded DNA obtained between amplification rounds in Illumina sequencing
strands are denatured
what happens after clusters are generated in Illumina sequencing
fluorescently labelled ‘reversible terminator’ bases are added to amplified single strands
how is the sequence obtained in Illumina sequencing
the signal from terminators is recorded, label is removed from terminator and cycle repeated
what does PacBio sequencing use
‘SMRT’ (single molecule real-time): an immobilized DNA polymerase and fluorescently tagged nucleotides
what does Oxford Nanopore sequencing use
an immobilized protein pore which passes single stranded DNA
how does Oxford Nanopore sequencing detect nucleotides
detects changes in current through pore which is dependent on the type of nucleotides in the pore
how does normal Nanopore sequencing work
single stranded DNA is passed through a membrane-bound pore and ion current is measured; there are no fluorescent labels
how does Pyrosequencing work (454)
it uses luciferase to detect pyrophosphate and produce light
in general are short read or long read sequencing methods more accurate
short read
