Different Platforms of sequencing ... Part IV

SOLiD Sequencing:

Step 1 : Library Preparation

Preparation of the two types of libraries such as fragment library or mate-paired library.Mate-paired libraries are libraries where two fragments of sequences are jointed with adapter and are known distant apart in the target.

Step 2 : Emulsion PCR/Bead Enrichment

Clonal bead populations in microreactors are prepared with template, PCR reaction components, beads, and primers. After PCR the templates were deanatured and bead enrichment process goes on to separate beads with extended templates from undesired beads. The template on the selected beads undergoes a 3’ modification to allow covalent attachment to the slide.

Step 3 : Bead Deposition

Deposit 3` modified beads onto a glass slide (Figure 1). During bead loading, deposition chambers enable you to segment a slide into one, four, or eight sections. A key advantage of the system is the ability to accommodate increasing densities of beads per slide, resulting in a higher level of throughput from the same system.

                                  Figure 1 : Attachment of beads in glass slide

Step 4 : Sequencing by Ligation

Primers hybridize to the P1 adapter sequence on the templated beads (Figure 2).

A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction.

Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length.

Following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles.

                                              

                                       Figure 2 : Sequencing by Ligation

Step 5 : Primer Reset

Five rounds of primer reset are completed for each sequence tag (Figure 3). Through the primer reset process, virtually every base is interrogated in two independent ligation reactions by two different primers. 

For example, the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1. This dual interrogation is fundamental to the unmatched accuracy characterized by the SOLiD System.

                                             Figure 3 : Primer Reset

Different platforms of Sequencing...Part III

Solexa/illumina Sequencing:

Solexa/illumina also used the technique : Sequencing by synthesis (SBS) with four fluorescentlylabeled nucleotides to sequence short DNA fragments. In compare to 454 platform this Solexa technique labeled deoxynucleoside triphosphate (dNTP) also act as a terminator for polymerization. After each dNTP incorporation, the fluorescent dye is imaged to identify the base and then enzymatically cleaved to allow incorporation of the next nucleotide. Since all four reversible terminator-bound dNTPs (A, C, T, G) are present as single separate molecules, natural competition minimizes incorporation bias.

Step A: Sample/Library preparation

The DNA sample of interest is sheared by focussed acoustic wave with a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-300bp are isolated via gel extraction and amplified using limited cycles of PCR using P5 and P7 primer (Figure 1).

Figure 1a: Amplification of Sequence of interest with P5 and P7 primers

Step B: Cluster Amplification:

The flow cell surface ( Figure: 1b) , a special type of plates for amplification of desired fragments, is coated with single stranded short oligonucleotides that are complemented to the sequences of the ligated adapters  during the sample preparation. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell channels and are exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface.

Figure 1b: The Flow cell surface 

Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in  "cluster station", an automated flow cell processor. Figure 2-7 illustrate stepwise amplification process of cluster.

Figure 2-7: Stepwise illustration of cluster amplification

Step C: Sequencing

A flow cell containing millions of unique clusters is then loaded into the illumina sequencer for automated cycles of extension and imaging.

The first cycle of sequencing starts by adding four labeled reversible terminators, primers, and DNA polymerase and after the first incorporation of a single fluorescent nucleotide, high resolution imaging of the entire flow cell produce the signal. Any signal above background identifies the physical location of a cluster, and the fluorescent emission identifies which of the four bases was incorporated at that position.

This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. Figure 8-13 illustrate stepwise sequencing process.

Figure 8-13: Stepwise illustration of Sequencing process

The output format of this platform is fastq .NGS assembler like SOAP de-novo , ALLPATHS-LG, Celera Assembler(wgs-assembler), Phrap, ABySS,CLCBio can be used to analysis and assemble data of illumina platform. 

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.