Next Gen Sequencing Flashcards
What is a nucleotide/ base composed off ?
Pentose sugar
Nucleotide Base
Triphosphate
What are the stages of PCR?
3 stage process
– Denaturation
– Anneal primer
– Extend new strand by incorporating dNTPs
uses thermostable DNA polymerase
Explain Sanger Sequencing
Samples are added into a mixture with regular nucleotides and dideoxynucleotides which are labelled and have terminators, fragments are gathered based on length and labelled nucleotides.
- DNA extracted and fragmented
- Fragments are cloned into vectors using restriction endonucleases.
- Sequence the gene library
- Assemble fragments
What are the limitations with Sanger Sequencing?
– Expensive, low throughput
– Labour intensive
– Low sensitivity – e.g. detection of mutations in
cancer need to be present in >30% of cells
What is mechanical DNA shearing? What are the two main methods?
DNA fragmentation. Methods:
* Sonication
- highly controllable
- shears DNA to desired lengths
- 150 bp - 75 Kb
- G-Tube (force DNA in centrifuge and through tiny aperture which breaks it into small pieces)
- centrifugal force
- fragment sizes between 6Kb to 20Kb
what is NEXT GENERATION SEQUENCING (NGS) ?
Technologies that enable you to sequence millions to billions of short sequences in a single run
Parallel sequencing either together or single molecule
Cheaper
How does 454 Next Generation Sequencing work?
- DNA is sheared into 300-800 bp fragments,
and the ends are “polished” by removing any unpaired
bases at the ends. - DNA is made into SSdna. Adapters are added to each end one containing biotin, which binds to a streptavidin-coated bead. (ratio of beads to DNA molecules is controlled so that most beads get only a single DNA attached to them)
- Oil is added to the beads and an emulsion is created. PCR
is then performed, Each bead ends up coated with about
a million identical copies of the original DNA. - Oil is removed beads are put into a “picotiter” plate, one bead per well.
- Pyrosequencing enzymes are attached to much smaller beads, which are then added to each well.
- Plate is then repeatedly washed with the each
of the four dNTPs, plus other necessary reagents, in
a repeating cycle. - Plate is coupled to a fibre optic chip. A CCD
camera records the light flashes from each well.
What are the limitations with 454 sequencing?
- no longer throughput enough ’only’ 1 million reads per run
- homopolymer (eg. AAAAA) issue is a big
problem A vs AA bases: 100% difference
Explain Illumina Enzymatic DNA shearing
Transposome (enz) loaded with loads of known DNA – cuts DNA and adds adaptors at same time – this is TAGMENTATION.
Known sequences act as priming sites for PCR reaction
What is sample pooling and its advantages and disadvantages?
A screening approach that combines samples from some number of people into one test.
- ADV – quick turnaround time, low cost
- DISADV – reduced to read number per sample
Explain the process of Illumina Sequecing
- Transposomes fragment DNA. 200 to 600 bp.
- Add adaptors with complementary sequences allow the DNA fragments to bind to the flow cell.
- Libraries loaded onto a flow cell and placed on the sequencer. The clusters of DNA fragments are amplified in a process called cluster generation, resulting in millions of copies of single-stranded DNA.
- Nucleotides bind to the DNA. Each nucleotide contains a fluorescent tag and a reversible terminator that blocks incorporation of the next base.
- After reading the forward DNA strand, the reads are washed away, and the process repeats for the reverse strand. This method is called paired-end sequencing.
what is paired end sequencing?
After reading the forward DNA strand, the process repeats for the reverse strand. This method is called paired-end sequencing.
advantages to paired end reading
- Enables better coverage uniformity
- insertion and deletion events can be detected by searching for reads that
have an unusual distance between their pairs - required for discovery of genome variation
Describe the preperation steps for PacBio
- Fragment DNA
- repair DNA damage on ends of fragmented DNA
- Add on smart bells/hairpins - form cicular piece of DNA
- Then add primers to circular piece of DNA – polymerase based so can extend DNA.
what is the role of Zero mode waveguides?
ZMW to distinguish the ideal fluorescent signal from the strong fluorescent backgrounds caused by unincorporated free-floating nucleotides
Why Pacbio real time sequencing?
A
PacBio use triphosphate linked fluorophore reduce steric hindrance, instead of fluorophore being linked to bases they are reversibly linked to triphosphates.
What are the advantages of Pacbio?
- Short waiting time for result and simple
workflow
– Generate basecalls in <1 day - long reads
- no amplification required
Explain the principle of Oxford Nanopore?
DNA is sequenced by passing through a microscopic pore base. It can be distinguished by how they effect ions flowing through the pore.
Explain the steps for Oxford nanopore?
Strand sequencing by passing DNA libraries through
protein nanopores inserted into a synthetic polymer
membrane.
1.One protein Unzips DNA
A second protein creates a pore in the membrane and hods an adaptor molecule
Flow of ions through pore creates current, bases block this differently for each base
Adapter molecule holds bases in place long enough for them to be identified
What is the role of SMRT Cell
which contains millions of tiny wells called zero-mode waveguides (ZMWs). Single molecules of DNA are immobilized in these wells, and as the polymerase incorporates each nucleotide, light is emitted, and nucleotide incorporation is measured in real time
How do Zero-mode waveguides (ZMWs), work within Pacbio?
Metallic nano structures within the ZMW, a molecule of DNA polymerase and the template DNA that is to be sequenced, is adsorbed to the bottom of the base, and nucleotides with fluorescent molecules attached are released into the ZMW chamber.
