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.