Different platforms of Sequencing...Part II

454 Pyrosequencing:

454 Sequencing uses a large-scale parallel pyrosequencing system capable of sequencing roughly 400-600 megabases of DNA per 10-hour run on the Genome Sequencer FLX with GS FLX Titanium series reagents.Fig 1 shows an overview how this platform works. 

Fig 1: Overview of 454 Pyrosequencing system by Roche

Step A: 454 Platform can sequence from genomic DNA, PCR products, BACs to cDNA.This DNA is mechanically sheared into fragments of a few hundred bp. 

Step B: 

• Library Adaptors are ligated to the fragments for use in subsequent steps (Figure 2).
• Library to DNA is then attached to Capture Beads by adaptors. Each bead carries a unique single-stranded library fragment. 
• Beads are then emulsified with amplification reagents in a water-in-oil mixture to trap individual beads in amplification microreactors.

 

 

Fig 2: Library Adaptors Ligation to DNA fragments

• Using emPCR millions of copies of each clone is generated using adaptor specific primer and attached to each bead.Before and after the amplification the bead looks like Figure 3.

Fig 3: Beads of emPCR

Step C and D: Amplified beads are then loaded to PicoTiterPlate, a specialized plate designed to load one bead per well. Therefore after sequencing one read data is generated for each bead. Tiny beads are loaded over each well to make it compact.

Step E: Individual nucleotides are flowed in sequence across the wells. Each incorporation of a nucleotide complementary to the template strand results in a chemiluminescent light signal recorded by the camera. The output format of this platform is SFF (Standard File Format) .NGS assembler like gsAssembler (Newbler), CLCBio, Celera Assembler(wgs-assembler), Phrap can be used to analysis and assemble data of 454 platform. Among them gsAssembler and Celera gives the best output for 454 data.

Now let's have a look of the pyrosequencing technique ........

Pyrosequencing is said to be "Sequencing by synthesis" compare to Sanger sequencing which exploit the principle of synthesis termination. It relies on the detection of pyrophosphate release on nucleotide incorporation during synthesis.

Pyrosequencing synthesize the complementary strand of a ssDNA in order to sequence it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.

• ssDNA is hybridized with compatable primer and incubated with DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.
• One of the four deoxynucleoside triphosphates (dNTPs) (dATPαS, is used instead of dATP as dATP may act as a substrate for luciferase enzyme) is added sequentially. 
• DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically which means the intensity of the light emitted is proportionate to the number of bases added.For example, the intensity of emitted light will be thrice if DNApol added three same bases consecutively compare to adding a single base. 

Fig 4: Pyrosequencing

• ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program (Figure 4).
• Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.