05. Pyrosequencing 454 Roche technology

Pyrosequencing is considered to be one of the first of the second-generation sequencing technologies. It was commercialized through Roche's 454 sequencing instrument, and allowed scientists to garner large amounts of sequencing data in a single run.

Unlike polony sequencing, pyrosequencing falls under sequencing by synthesis, meaning the sequence is resolved while forming the sample's complementary strand. However, similar to polony sequencing, pyrosequencing uses emulsion PCR.

At its core, the pyrosequencing technique relies on the detection of pyrophosphate molecules that are released during DNA synthesis. This allows for the generation of light, which is then detected by a sensor.

What is Pyrophosphate?

In your typical dNTP molecule, there are three phosphate that are attached to the 5' carbon of the deoxyribose sugar. The first (which is attached to the sugar) is called the α-phophate. The next is β-phosphate and the last is γ-phosphate.

During replication, the α-phosphate of each incoming complementary nucleotide is joined enzymatically by a phosphodiester linkage to the 3'-OH group of the last nucleotide in the growing strand.

During this reaction, the β- and γ-phosphates are cleaved off in a unit called the pyrophosphate (PPi).

Structure of deoxynucleoside triphosphate dNTP
Pyrophosphate is a by-product of DNA elongation.

Procedure

In second generation DNA sequence techniques, a cycle is established to resolve each nucleotide. Here is the cycle used in pyrosequencing:

1) Emulsion PCR

After emulsion PCR is performed, each enriched bead is placed in one of the many picoliter-volume wells of the sequencing machine.

2) Adding in a dNTP

One of the four dNTP's is added. If the next sequence of the growing strand is complementary to the dXTP, PPi is released.

3) PPi causes light to be released

PPi reacts with ATP sufurylase, generating ATP. This reacts with luciferase to produce light. The flash of light is recorded by a camera - intensity is proportional to more dXTP's being added.

Here is the chemical reaction that takes place to generate light.

Reaction chemistry to Pyrosequencing.
Steps to detecting a nucleotide by pyrosequencing: 1) PPi is generated if dXTP is incorporated. 2) PPi reacts and through a series of reactions, light is generated. 3) Light gets picked up by the sensor. 4) The more intense the light is, the more dXTP's are in that region of sequence.

4) Remaining reagents washed away

Any remaining deoxynucleoside triphosphate (dXTP) and ATP are degraded by apyrase and washed away.

5) Repeat in a cyclic fashion

This process is repeated from step 2 until all bases are sequenced.

Sequencing Machines

The sequencing machines out in industry that use pyrosequencing includes Roche's 454 platform.

Two of Roche's DNA sequencing instruments.
Roche GS Junior+ and GS FLX Titanium XL+
GS Junior+ GS FLX Titanium XL+ GS FLX Titanium XLR70
Bases per run ~100,000 ~1,000,000 shotgun ~1,000,000 shotgun
Read Length ~700 bp Up to 1,000 bp Up to 600 bp
Mode Read Length 700 bp 700 bp 450 bp
Run time 18 hours 23 hours 10 hours
Consensus Accuracy 99% 99.997% 99.995%

Pros and Cons

Some good points to pyrosequencing is the long read sizes, and fast run times. However, runs are expensive, and the homopolymer errors are frequent due to a low sensitivity.

Videos

References

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