Next generation 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.
Similar to PCR but only uses a single primer and polymerase.
- DNA extracted and fragmented
- Fragments are cloned into vctors 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
Explain how 454 Next Generation Sequencing works?
- DNA is sheared into 300-800 bp fragments,
and the ends are “polished” by removing any unpaired
bases at the ends.
2.DNA is made ssdna, Adapters are added to each end one containing biotin, which binds to a streptavidincoated 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.
4.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 fiber 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 - AAAAA vs AAAAAA bases: 20% difference
Explain illumina Enzymatic DNA shearing?
- Transposomes fragement and tag DNA sequence and insert
a- Locus specific primer F/R ( Bind to target DNA to allow specific amplification )
b- index 1 & 2 ( – 8bp DNA sequence that is unique for each sample, used for alligning reads to samples)
c - P5/7 tail ( Bind Product to flow cell)
What is sample pooling and its advantages and disadvantages?
a screening approach that combines samples from some number of people into one test.
Advantages
– Reduces reagent cost
– Quicker turnaround time per sample
Disadvantages
– Reduced read number per sample
– Introduces normalisation step to minimise variation in
read number per sample
Explain the process of Illumina sequencing?
- Break up the DNA using transposomes? into more manageable fragments of around 200 to 600.
- Add adaptors (Locus specific primer F/R, Index 1 & 2, P5/7 tail)
- DNA is made ssDNA and washed over flow cell via P5/7 tail
- Replicated to form small clusters of DNA with the same sequence (many clusters = stronger signal)
- Unlabelled nucleotide bases? and DNA polymerase? are then added to lengthen and join the strands of DNA attached to the flowcell = double stranded DNA
6.Double-stranded DNA is then broken down into single-stranded DNA using heat
7.Primers and fluorescently labelled terminators are added one at a time and Lasers are passed over the flowcell to activate the fluorescents that is recorded by a camera.
( No more bases can be added after terminator so one a time means you know which base and the signal is strong due to number of strands)
Explain Paired end indexed sequencing and its benifits?
In single-end reading, the sequencer reads a fragment from only one end to the other, generating the sequence of base pairs. In paired-end reading it starts at one read, finishes this direction at the specified read length, and then starts another round of reading from the opposite end of the fragment.
- Enables better coverage uniformity by allowing highly repetitive sequence
to be anchored by unique paired read - 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
What is Mechanical DNA shearing, describe the principles of the 2 main methods?
Sonication
* Highly controllable
* Shears DNA to desired
lengths (150bp-75Kb)
* Multi-sample parallelprocessing (96 samples)
G-Tube
* Centrifugal force
* Fragments sizes ranging
from 6kb to 20kb
* Low throughput (12
samples
What is the principle behind PacBoi sequencing use of The zero-mode waveguide?
PacBio SMRT Sequencing use ZMW to distinguish the ideal fluorescent signal from the strong fluorescent backgrounds caused by unincorporated free-floating nucleotides.
The binding of a DNA polymerase and the template DNA strand is anchored to the bottom glass surface of a ZMW. Laser light travels through the bottom surface of a ZMW and not completely penetrates it, since the ZMW dimensions are smaller than the wavelength of the light. Therefore, it allows selective excitation and identification of light emitted from nucleotides recruited for base elongation.
Describe the preperation steps for PacBio?
1.Determine the quality of genomic DNA (gDNA)
2.Shear gDNA using a g-TUBE (Covaris)
3.Select size and adjust concentration
4.Repair DNA damage and ends of fragmented DNA
5.Conduct DNA purification
6.Blunt-end ligation using blunt adapters - A SMRTbell™ template is a double-stranded DNA template capped by hairpin loops at both ends
7.Purify template for submission to a sequencer
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, alongside 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.
