Illumina sequencing in details.........

Illumina basic steps: sample prep-----cluster generation on solid phase---sequencing by synthesis with reversible terminator--Data analysis
In Illumina splatform, sequencing occurs by synthesis. Sequencing by synthesis (SBS) technology that is the foundation of Illumina sequencing instruments.

Use of clonal arrays and massively parallel sequencing of short reads using solid phase sequencing by reversible terminators (the SBS) became the basis of a new DNA sequencing approach. A fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias.

Reversible terminator sequencing differs from the traditional Sanger method in that, instead of terminating the primer extension irreversibly using dideoxynucleotide, modified nucleotides are used in reversible termination. Whilst many other techniques use emulsion PCR to amplify the DNA library fragments, reversible termination uses bridge PCR, improving the efficiency of this stage of the process.

Reversible terminators can be grouped into two categories: 3′-O-blocked reversible terminators and 3′-unblocked reversible terminators.Only the correct base anneals to the target and is subsequently ligated to the primer. The solid support is then imaged and nucleotides that have not been incorporated are washed away and the fluorescent branch is cleaved using TCEP (tris(2-carboxyethyl)phosphine). TCEP also removes the 3’-O-azidomethyl group, regenerating 3’-OH, and the cycle can be repeated 

(https://www.slideshare.net/cosentia/illumina-gaiix-for-high-throughput-sequencing)

The amplification of single DNA molecules into clusters enhanced the fidelity and accuracy of gene calling, also reducing the cost of the system optics through generation of a stronger signal.

Using this technology, in 2005, bacteriophage phiX-174 genome was sequenced

The SBS technology generated large quantity of sequence data (3 million bases) from a single run (multiple times higher than that of Sanger data). Now, Illumina SBS technology-based instruments can generate multiple terabases (Tb) of data per run.

Diff illumina sequencers: MiniSeq, MiSeq, NextSeq, HiSeq

USes: Large Whole-Genome Sequencing (human, plant, animal), Small Whole-Genome Sequencing (microbe, virus), Exome Sequencing, Targeted Gene Sequencing (amplicon, gene panel), Whole-Transcriptome Sequencing, Gene Expression Profiling with mRNA-Seq, Targeted Gene Expression Profiling, miRNA & Small RNA Analysis, DNA-Protein Interaction Analysis, Methylation Sequencing, 16S Metagenomic Sequencing

Sample preparation

Randomly fragment genomic DNA and ligate adapters to both ends of the fragments.

 Figure 3: Attach DNA to Surface

Cluster Generation

Several samples can be loaded onto the eight-lane flow cell

Sequencing templates are immobilized on a proprietary flow cell surface. Enzyme is applied.

Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity. Ten million single-molecule clusters per square centimeter are achieved.

Sequencing by Synthesis

Sequencing by SBS technology uses four fluorescently labeled nucleotides to sequence the tens of millions of clusters on the flow cell surface in parallel

During each sequencing cycle, a single labeled deoxynucleoside triphosphate (dNTP) is added to the nucleic acid chain.

The nucleotide label serves as a terminator for polymerization, so after each dNTP incorporation, the fluorescent dye is imaged to identify the base and then 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

(http://www.atdbio.com/content/58/Next-generation-sequencing)

Base calls are made directly from signal intensity measurements during each cycle, which greatly reduces raw error rates compared to other technologies.

Robust base calling in repetitive sequence regions and within homopolymers is ensured.

Analysis

Deep sampling and uniform coverage isis used to generate a consensus

Illumina data collection software enables to align sequences to a reference

#TruSeq family of reagents, base calls above Q30, generate several gigabases of DNA sequence per run

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Sample prep: 2h
Load sample
Sequence button-experiment name
Load flow cell---click continue
Single cartridge contains clustering, sequencing, PE reagents (reagent kit)
RFID-based tracking
Put PR2 bottle
Variant reporting, on-instrument less than 2 hrs, base calls and quality values in industry standard formats, real time run monitoring

If error shown, exit out of run setup. Return to home screen. Unload all consumables. Restart run setup. Reload all consumables.


To resolve issue, email Technical Support at techsupport@illumina.com and provide the affected lot number of the bottle.