New sequencing technology Flashcards
What are some limitations of the Sanger method? List 5.
- Maximum read length is approx. 1000 bases
- High cost per base
- Long to set up
- High DNA conc. needed
- Some regions still don’t sequence
What is the principle of the ‘sequencing in synthesis’ method?
Target DNA is replicated. Replicates are made single-stranded and fixed. Sequencing occurs using a DNA polymerase based method.
What are the 6 steps for GS FLX by Roche 454?
- Double-stranded DNA broken up into fragments using a nebuliser
- DNA is polished
- 2 adapters, A and B, are ligated to each end of the fragment
- A biotin tag on the B adapter forms a strong bond to Streptavidin beads
- DNA denatured and split into single strands
- These are used as templates in emPCR
What does it mean when the DNA is polished?
It is made blunt-ended.
How long are both adapters?
~44bp.
On which end of the B adapter is the biotin tag?
The 5’ end.
What are the capture beads covered in?
Oligonucleotides (short nucleotide polymers) that are complementary to the B adapters.
How big are the streptavidin capture beads?
Approx. 30 micrometers
What is emPCR?
Emulsion PCR.
How does emPCR work?
- PCR reagents plus the streptavidin beads covered in DNA fragments are added to emulsion oil
- Water droplets form micro reactors around the PCR reagents and beads
- Each micro reactor undergoes a PCR reaction
- This results of a clonal amplification of the single DNA fragments. These attach the oligonucleotides on the beads.
- Beads are then removed from the emulsion
After emPCR pyrosequencing occurs. What happens?
- The amplified DNA is made single-stranded and a sequencing primer is added.
- The beads are loaded into wells in a PicoTiter plate along with enzyme beads
- Nucleotides are flowed sequentially across the plate
- When a DNA base in incorporated into the sequence by DNA polymerase a fluorescent signal is emitted, which is detected by a camera
How many capture beads fit into each well of a PicoTiter plate?
1.
What enzyme beads also go into the wells?
Luciferase and Sulfurylase
Why are the different nucleotides flowed sequentially?
The same fluorescent signal is produced for each nucleotide.
What is the name for the flow of nucleotides across the PicoTiter plate?
A nucleotide wash.
What produces the fluorescent signal?
Luciferase
Each well produces its own flow gram. What does this represent?
The sequence of nucleotides in the fragment.
How is the sequence from the whole genome produced from flow grams?
The sequences of individual fragments are compared to a reference sequence to help assemble them in order.
What are 2 advantages of 454 GS FLX?
- Some machines can sequence up to 30GB per run
2. It is cheaper than Sanger sequencing
What are 3 disadvantages of 454 GS FLX?
- Cannot sequence long homopolymer regions
- Cost per run is still expensive
- Higher error rate than Sanger sequencing
Thus which 3 processes make up a whole run of 454 GS FLX?`
DNA capture using beads, emPCR then pyrosequencing using a PicoTiter plate.
What initial steps does Illumina sequencing have in common with 454?
- Break target DNA up into fragments
- Attach adaptors to each end
- Make fragments single stranded
How are DNA fragments then amplified in Illumina sequencing? Give 7 steps.
- ssDNA fragments attached to a flow cell
- Flow cell is covered in millions of primers complementary to adapters
- Fragments anneal to flow cell leaving one free adapter at the end
- Free adapter also binds to complementary primer, creating a bridge
- Add PCR reagents so complementary strand is synthesised to form a double-bridge
- Separate the double bridges so now each complementary strand is joined to cell at one end, free adapter sticking up.
- Repeat this bridge building/breaking process to form clusters of clonally amplified DNA on the flow cell
What happens after clonal amplification in Illumina sequencing?
Remove the strands that are joined to flow cell by one type of adapter. Leaves DNA fragment clusters that are joined by the other adapter. These act as sequencing templates. Add complementary sequencing primers.
Reversible terminators are added in Illumina sequencing. What are they and why are they used?
Chemically modified nucleotides that, when incorporated onto a template, prevent further addition of nucleotides until the chemical modification is removed.
What happens to reversible terminators when excited by a laser?
They release fluorescent signals specific to each nucleotide.
What happens when the reversible terminators are added?
All 4 reversible terminators are added simultaneously and bind to the first free nucleotide in a template. Each cluster of templates will have a different nucleotide in the first free position. The flow cell is then exposed to a laser.
What happens after laser excitation in Illumina sequencing?
Each cluster will fluoresce differently (i.e. different colours) depending on which reversible terminator was added. A picture is taken.
After the first exposure to a laser, what happens in Illumina sequencing?
The steps are repeated: the chemical modification of the bound reversible terminator is removed, allowing another one to be added. The sample is then exposed to a laser again. It is in this way we can begin to sequence each fragment.
How then are the random short sequences of Illumina sequencing assembled?
They are compared to a reference sequence.
In Illumina sequencing, how many samples can each flow cell support?
8.
The sequence fragments for Illumina are shorter than those for 454. How long are they?
~30-45bp
Give 5 advantages of Illumina.
- No issue with homopolymers as sequencing is base by base position
- Simultaneous base addition reduces misincorporation rate
- Cheaper than 454
- Can sequence up to 200GB per run
- Requires less initial start DNA
Give 1 disadvantage of Illumina.
Error rate still greater than Sanger.
What similarities are there between 454 and SOLid sequencing by ABI?
The initial steps are the same: DNA is fragmented, adapters added, capture beads are used, emPCR clonally amplifies fragments, beads are removed from emulsion.
When does SOLid sequencing begin to differ from 454?
Beads are deposited directly
onto glass slide, instead of a PicoTiter plate.
How many chambers is the slide divided up into?
8.
How is sequencing done in SOLid?
By ligation: primers hybridise to adapters on the beads. 4 fluorescently labelled di-base probes compete for ligation to the primer. This sequences base position ‘n’ in the fragment.
How is the whole fragment sequenced in SOLid?
The extension product is removed after each ligation cycle and the template reset with a different primer, complementary to n-1 in the sequence.
What determines the final read length in SOLid?
The number of ligation cycles.
In SOLid, the colour (from a di-base probe) in a reading DOES NOT indicate a base. What does each reading tell you?
Information from 2 base positions.
How do you decode the readings from SOLid?
You need to know one base in the sequence. We always know this: base 0 is the adapter. You compare the readings to a chart that tells you, if you know the colour and first base position, what the second base must be.
How long are the fragments sequenced in SOLid?
20-25bp. These are aligned to a reference as in 454 and Illumina.
Give 3 advantages of SOLid?
- No problems with homopolymer regions, base-by-base sequencing.
- Greatly reduced error rate as 2 bases need to be known for sequencing
- Can sequence up to 6GB per run
Give 2 disadvantages of SOLid.
- Most expensive of the 3
2. Very short sequencing reads of 30-40bp
True Single Molecule Sequencing is very similar to Illumina and 454. What are the differences?
- No PCR step (only uses a single DNA molecule)
2. Requires a heliscope
You can sequence via hybridisation in microarrays. What do you need?
A reference sequence. Basically multiple DNA fragments on a chip each with a different nucleotide at the centre. Genomic DNA added and hybridisation depends on which nucleotide is in the centre of the fragment.
What does nanopore sequencing need?
Electricity to drive DNA through a nanopore. The nanopore changes state depending on which nucleotide is forced through.
What is real time DNA sequencing?
Basically you get a single polymerase molecule and some fluorescently labelled dNTPs and watch it assemble a complementary strand to a template…
What does visualisation in real time sequencing require?
A zero-mode waveguide: basically condenses light energy. Fluorophores added to the terminal phosphate of the dNTPs absorbs then re-emits light.
