New sequencing technology

Question 1

What are some limitations of the Sanger method? List 5.

Answer 1

1. Maximum read length is approx. 1000 bases
2. High cost per base
3. Long to set up
4. High DNA conc. needed
5. Some regions still don’t sequence

Question 2

What is the principle of the ‘sequencing in synthesis’ method?

Answer 2

Target DNA is replicated. Replicates are made single-stranded and fixed. Sequencing occurs using a DNA polymerase based method.

Question 3

What are the 6 steps for GS FLX by Roche 454?

Answer 3

1. Double-stranded DNA broken up into fragments using a nebuliser
2. DNA is polished
3. 2 adapters, A and B, are ligated to each end of the fragment
4. A biotin tag on the B adapter forms a strong bond to Streptavidin beads
5. DNA denatured and split into single strands
6. These are used as templates in emPCR

Question 4

What does it mean when the DNA is polished?

Answer 4

It is made blunt-ended.

Question 5

How long are both adapters?

Answer 5

~44bp.

Question 6

On which end of the B adapter is the biotin tag?

Answer 6

The 5’ end.

Question 7

What are the capture beads covered in?

Answer 7

Oligonucleotides (short nucleotide polymers) that are complementary to the B adapters.

Question 8

How big are the streptavidin capture beads?

Answer 8

Approx. 30 micrometers

Question 9

What is emPCR?

Answer 9

Emulsion PCR.

Question 10

How does emPCR work?

Answer 10

1. PCR reagents plus the streptavidin beads covered in DNA fragments are added to emulsion oil
2. Water droplets form micro reactors around the PCR reagents and beads
3. Each micro reactor undergoes a PCR reaction
4. This results of a clonal amplification of the single DNA fragments. These attach the oligonucleotides on the beads.
5. Beads are then removed from the emulsion

Question 11

After emPCR pyrosequencing occurs. What happens?

Answer 11

1. The amplified DNA is made single-stranded and a sequencing primer is added.
2. The beads are loaded into wells in a PicoTiter plate along with enzyme beads
3. Nucleotides are flowed sequentially across the plate
4. When a DNA base in incorporated into the sequence by DNA polymerase a fluorescent signal is emitted, which is detected by a camera

Question 12

How many capture beads fit into each well of a PicoTiter plate?

Answer 12

1.

Question 13

What enzyme beads also go into the wells?

Answer 13

Luciferase and Sulfurylase

Question 14

Why are the different nucleotides flowed sequentially?

Answer 14

The same fluorescent signal is produced for each nucleotide.

Question 15

What is the name for the flow of nucleotides across the PicoTiter plate?

Answer 15

A nucleotide wash.

Question 16

What produces the fluorescent signal?

Answer 16

Luciferase

Question 17

Each well produces its own flow gram. What does this represent?

Answer 17

The sequence of nucleotides in the fragment.

Question 18

How is the sequence from the whole genome produced from flow grams?

Answer 18

The sequences of individual fragments are compared to a reference sequence to help assemble them in order.

Question 19

What are 2 advantages of 454 GS FLX?

Answer 19

1. Some machines can sequence up to 30GB per run

2. It is cheaper than Sanger sequencing

Question 20

What are 3 disadvantages of 454 GS FLX?

Answer 20

1. Cannot sequence long homopolymer regions
2. Cost per run is still expensive
3. Higher error rate than Sanger sequencing

Question 21

Thus which 3 processes make up a whole run of 454 GS FLX?`

Answer 21

DNA capture using beads, emPCR then pyrosequencing using a PicoTiter plate.

Question 22

What initial steps does Illumina sequencing have in common with 454?

Answer 22

1. Break target DNA up into fragments
2. Attach adaptors to each end
3. Make fragments single stranded

Question 23

How are DNA fragments then amplified in Illumina sequencing? Give 7 steps.

Answer 23

1. ssDNA fragments attached to a flow cell
2. Flow cell is covered in millions of primers complementary to adapters
3. Fragments anneal to flow cell leaving one free adapter at the end
4. Free adapter also binds to complementary primer, creating a bridge
5. Add PCR reagents so complementary strand is synthesised to form a double-bridge
6. Separate the double bridges so now each complementary strand is joined to cell at one end, free adapter sticking up.
7. Repeat this bridge building/breaking process to form clusters of clonally amplified DNA on the flow cell

Question 24

What happens after clonal amplification in Illumina sequencing?

Answer 24

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.

Question 25

Reversible terminators are added in Illumina sequencing. What are they and why are they used?

Answer 25

Chemically modified nucleotides that, when incorporated onto a template, prevent further addition of nucleotides until the chemical modification is removed.

Question 26

What happens to reversible terminators when excited by a laser?

Answer 26

They release fluorescent signals specific to each nucleotide.

Question 27

What happens when the reversible terminators are added?

Answer 27

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.

Question 28

What happens after laser excitation in Illumina sequencing?

Answer 28

Each cluster will fluoresce differently (i.e. different colours) depending on which reversible terminator was added. A picture is taken.

Question 29

After the first exposure to a laser, what happens in Illumina sequencing?

Answer 29

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.

Question 30

How then are the random short sequences of Illumina sequencing assembled?

Answer 30

They are compared to a reference sequence.

Question 31

In Illumina sequencing, how many samples can each flow cell support?

Answer 31

8.

Question 32

The sequence fragments for Illumina are shorter than those for 454. How long are they?

Answer 32

~30-45bp

Question 33

Give 5 advantages of Illumina.

Answer 33

1. No issue with homopolymers as sequencing is base by base position 2. Simultaneous base addition reduces misincorporation rate 3. Cheaper than 454 4. Can sequence up to 200GB per run 5. Requires less initial start DNA

Question 34

Give 1 disadvantage of Illumina.

Answer 34

Error rate still greater than Sanger.

Question 35

What similarities are there between 454 and SOLid sequencing by ABI?

Answer 35

The initial steps are the same: DNA is fragmented, adapters added, capture beads are used, emPCR clonally amplifies fragments, beads are removed from emulsion.

Question 36

When does SOLid sequencing begin to differ from 454?

Answer 36

Beads are deposited directly | onto glass slide, instead of a PicoTiter plate.

Question 37

How many chambers is the slide divided up into?

Answer 37

8.

Question 38

How is sequencing done in SOLid?

Answer 38

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.

Question 39

How is the whole fragment sequenced in SOLid?

Answer 39

The extension product is removed after each ligation cycle and the template reset with a different primer, complementary to n-1 in the sequence.

Question 40

What determines the final read length in SOLid?

Answer 40

The number of ligation cycles.

Question 41

In SOLid, the colour (from a di-base probe) in a reading DOES NOT indicate a base. What does each reading tell you?

Answer 41

Information from 2 base positions.

Question 42

How do you decode the readings from SOLid?

Answer 42

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.

Question 43

How long are the fragments sequenced in SOLid?

Answer 43

20-25bp. These are aligned to a reference as in 454 and Illumina.

Question 44

Give 3 advantages of SOLid?

Answer 44

1. No problems with homopolymer regions, base-by-base sequencing. 2. Greatly reduced error rate as 2 bases need to be known for sequencing 3. Can sequence up to 6GB per run

Question 45

Give 2 disadvantages of SOLid.

Answer 45

1. Most expensive of the 3 | 2. Very short sequencing reads of 30-40bp

Question 46

True Single Molecule Sequencing is very similar to Illumina and 454. What are the differences?

Answer 46

1. No PCR step (only uses a single DNA molecule) | 2. Requires a heliscope

Question 47

You can sequence via hybridisation in microarrays. What do you need?

Answer 47

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.

Question 48

What does nanopore sequencing need?

Answer 48

Electricity to drive DNA through a nanopore. The nanopore changes state depending on which nucleotide is forced through.

Question 49

What is real time DNA sequencing?

Answer 49

Basically you get a single polymerase molecule and some fluorescently labelled dNTPs and watch it assemble a complementary strand to a template...

Question 50

What does visualisation in real time sequencing require?

Answer 50

A zero-mode waveguide: basically condenses light energy. Fluorophores added to the terminal phosphate of the dNTPs absorbs then re-emits light.