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.

Different platforms of Sequencing...Part I

Now a days commercially there are 4 types of Platform used for sequencing purpose.

• Sanger Sequencing
• 454 Pyrosequencing
• Solexa/illumina Sequencing
• SOLiD Sequencing

Among them the last 3 are used for high-throughput sequencing , so are the candidates for Genome Sequencing Projects.

Let's see first how the Sanger Sequencing works!!

Sanger Sequencing:

During 1974 two independent methods for Sequencing was invented initiating a new era of Molecular Biology named as Sanger Method and Maxam-Gilbert Method . Sanger Sequencing is also called dideoxy sequencing as here dideoxynucleotides are used along with normal nucleotides. This method is popular for it's simplicity ... the dideoxynucleotides acts as chain terminator as these bases don't have 3 ' OH group thus can't form the phosphodiester bond with upcoming nucleotide.

So here four types of dideoxynucleotides are used in 4 different tubes along with 4 types of normal nucleotide. Say in the G tube only dideoxyGuanine is added to normal A , T , C and G .

In Sequencing reaction the DNA sample should first be denatured by heat to get single strand. The primer is annealed at the 5 ' end and then the termination reaction starts in each tube. This amplified products are subjected for electrophoresis and are separated depending on fragment gradient. After electrophoresis the gel looks like something the picture bellow.

Now a day the 4 type of dideoxynucleotides are labelled with different dyes and can be differentiated by different emission spectra. So all the reactions can go on in a single tube with proper reaction condition in thermal cycle and thus can be subjected for electrophoresis in single lane instead of four!!

The image bellow shows a standard sequence chromatogram using Sequence Scanner by Applied Biosystem.

For the termination reaction Big dye reagent of Applied Biosystem is the most popular one with their many Capillary Sequencer. I have a plan to write about Capillary Electrophoresis Sequencing in details afterwards.

Many commercial institute provide the facility of Sanger sequencing. The output format of Sanger Sequence file is generally in Standard Chromatogram Format (.SCF) or ABI sequencer data files (.ABI and .AB1) or PHRED output files (.PHD). They can be assembled by various assembly software like DNA Dragon , Phrap , CLC Bio , etc. DNA Dragon is free for all and Phrap is only for academic purpose.