Next generation sequencing Flashcards
(26 cards)
2 key features of NGS that make it improved from sanger
- clonal amplification of DNA fragments using massively parallel PCR based techniques
- sequencing of all DNA clones produced in parallel using a light detection only method (rather than electrophoresis)
pyrosequencing
DNA sequencing technique based on detecting the release of pyrophosphate (PPi) during DNA synthesis, rather than fragment size as in Sanger sequencing
emulsion PCR
a type of PCR (Polymerase Chain Reaction) that happens inside tiny water droplets suspended in oil. Each droplet acts like a miniature test tube to amplify DNA fragments in a way that keeps each DNA template physically isolated
process of emulsion pcr followed by pyrosequencing
fragment genome into tiny pieces > ligate adaptors to ends of each fragment so primers can be designed > create DNA:water/oil emulsion in excess so 1 DNA per droplet > PCR > spread individual PCR reactions onto pico titer plates > pyrosequencing using primers complementary to adaptors
enzymes used in pyrosequencing
sulfurylase
luciferase
apyrase
sulfurylase
converts pyrophosphate into ATP
luciferase
produces light from ATP
The amount of light is directly proportional to the number of nucleotides incorporated.
apyrase
degrades unincorporated nucleotides
cons of pyrosequencing v sanger
higher error rate
not well suited for larger genome sequencing
illumina sequencing
bridge amplification on glass slide to yield clonal clusters of DNA
followed by reversible dye terminator chemistry
bridge amplification
DNA fragments are ligated with adapter sequences.
The flow cell is coated with two types of primers, each complementary to one of the adapters.
Each DNA molecule hybridizes (binds) to a primer on the flow cell surface.
The DNA bends over, forming a bridge.
The free adapter end binds to a nearby complementary primer on the flow cell.
DNA polymerase extends the strand, copying the DNA and anchoring the new strand to the flow cell.
The double-stranded DNA is denatured (separated into single strands).
The original strand is washed away, leaving the new strand bound to the surface.
This process repeats multiple times.
Each DNA fragment is amplified in place, forming a clonal cluster — thousands of identical copies of the same DNA sequence.
reversible dye terminator chemistry
each nucleotide has a unique fluorescent dye and a reversible terminator group that prevents further extension after incorporation.
only one nucleotide can be added to each DNA strand because of the terminator group.
DNA polymerase adds the complementary base to the template strand.
No more bases can be added until the terminator is removed.
A laser excites the fluorophores, and a camera takes an image of the flow cell.
Each clonal cluster emits light corresponding to the base that was added.
The color is read and recorded as that base.
The fluorescent dye and terminator are chemically removed.
The strand is now ready for the next cycle of base incorporation.
cycle repeated.
where on the nucleotide are the fluorophore and reversible terminator located
fluorophore is bound to the base
reversible terminator is bound to the 3 carbon of the sugar
newer NGS
no PCR needed/ no clones
sequence data obtained in real time
SMRT and nanopore sequencing
SMRT sequencing
Polymerase molecule attached to bottom of sequencing well
Illuminate well so that is just illuminates volume taken up by the polymerase. This part of the well is referred to as the zero-mode waveguide.
Fluorescently labelled dNTPs are only excited if they enter this region and polymerase incorporates them into chain and the fluorophore is released and detected
where us the fluorophore attached to the nucleotide in SMRT
a phosphate group so it is released when the nucleotide is incorporated (rather than using laser)
nanopore sequencing
DNA sequences in this method are ion channels embedded into an inert membrane and placed into electrolyte solution. pd applied and flow of ions through nanopore. obstruction through nanopore reduced in flow causing deflection in current
nanopore vs SMRT
higher throughput than SMRT
generates longer read lengths
motor enzyme in nanopore sequencing
interacts with nanopore and feeds DNA strand through
NGS in medical genetics
identify disease genes in complex genetic diseases > SNPs
GWAS, microarrays
microarray based SNP typing
oligonucleotide probes complementary to all known common SNP loci in human genomes spotted onto a microarray. labelled DNA hybridises onto microarray. laser scanning to identify SNPs
missing heritability
genetic contribution to complex diseases that we know must exist but were not identified in common variant GWAS
RVAS
rare variant hypothesis to find missing heritability
common variant hypothesis
GWAS
small amount of heritability identified
