Principle and Workflow of Illumina Next-generation Sequencing

Illumina, established in 1998 in San Diego, CA, is a leading company in the field of sequencing. In 2006, Illumina acquired Solexa, got the next-generation high-throughput sequencing technology and developed it into a mainstream technology on the market. It currently provides sequencing systems such as MiSeq, HiSeq 2500, HiSeq 3000, HiSeq 4000, HiSeq X Ten, HiSeq X five, NextSeq 550.

Illumina NGS applications

NGS has a very wide range of applications, it can be used for whole-genome sequencing, targeted region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis, helping people unlocking the power of the gene.

The core principle of Illumina NGS

The Illumina next-generation sequencing (NGS) method is based on sequencing-by-synthesis (SBS), and reversible dye-terminators that enable the identification of single bases as they are introduced into DNA strands.

Theoretically, Illumina NGS technology resembles Sanger sequencing. During the cyclic process of DNA synthesis sequencing, a DNA polymerase catalyzes the incorporation of fluorescently labeled deoxyribonucleotide triphosphates (dNTPs) onto a DNA template strand. Each cycle, as the dNTP is incorporated, the nucleotide is identified via fluorescence excitation. However, diverging from Sanger sequencing, which sequences the entire completed DNA segment, NGS has revolutionized the process through its high-throughput parallel sequencing method.

The workflow of Illumina NGS

Step 1. Library preparation

Through ultrasonic fragmentation, the genomic DNA becomes DNA fragment with 200-500bp in length. The 5′ and 3′ adapter are added to the two ends of these small segments, "tagmentation" combines the fragmentation and ligation reactions into single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified. The sequencing library is constructed.

Figure 1. The 1st step: library preparation

Step 2. Cluster generation

Flow cell is a channel for adsorbing mobile DNA fragments, and it’s also a core sequencing reactor vessel — all the sequencing happens here. The DNA fragments in the sequencing library will randomly attach to the lanes on the surface of the flow cell when they pass through it. Each flow cell has 8 Lanes, each lane has a number of adapters attached to the surface, which can match the adapters added at the ends of the DNA fragment in the building process, which is why flow cell can adsorb the DNA after the building, and can support the amplification of the bridge PCR on the surface of the DNA. In theory, there is no mutual influence between these lanes.

Bridge PCR was performed using the adapters on flow cell surface as template, after continuous amplification and mutation cycles, each DNA fragment will eventually be clustered in bundles at their respective locations, each containing many copies of a single DNA template.

The purpose of this process is to amplify the signal intensity of the base to meet the signal requirements for sequencing. When cluster generation is complete, those templates are ready for sequencing.

Figure 2. The 2nd step: cluster generation

Step 3. Sequencing

The sequencing method is based on sequencing-by-synthesis (SBS). DNA polymerase, connector primers and 4 dNTP with base-specific fluorescent markers were added to the reaction system. The 3′-OH of these dNTP are protected by chemical methods, which ensures that only one base will be added at a time during the sequencing process. All unused free dNTP and DNA polymerase are eluted after the synthesis reaction finished.

Then, buffer solution needed for fluorescence excitation are added, the fluorescence signal is excited by laser, and fluorescence signal is recorded by optical equipment.

Finally, the optical signal is converted into sequencing base by computer analysis. When the fluorescence signal is recorded, a chemical reagent is added to quench the fluorescence signal and remove the dNTP 3′-OH protective group, so that the next round of sequencing reaction can be performed.

Figure 3. The 3rd step: sequencing

Step 4. Alignment & Data analysis

The newly identified sequence reads are aligned to a reference genome, then many variations of bioinformatics analysis are possible such as SNP/InDel/SV/CNV calling, annotation and statistics, pathway enrichment analysis, population genetics analysis and more.

Figure 4. The 4th step: alignment and data analysis.

The above is Illumina NGS chemistry overview, the SBS technology allows single-end and two-end sequencing, improves the ability to fully identify any genome.

Advantages of Illumina NGS

High throughput: Illumina’s NGS technology has the capacity to generate an abundance of sequence data within a relatively short timespan.

High resolution: The advanced imagistic prowess of the Illumina sequencing platform enables accurate acquisition of sequential data of every added base, thus proffering precise information on base sequences.

Cost-efficiency: The affordability of the technology facilitates researchers in the successful implementation of large-scale sequencing projects.

Flexibility: The Illumina platform supports a wide range of sample types and library construction methods, making it suitable for various research objectives and experimental designs.

Fast pace: With Illumina’s NGS technology, large-scale sequencing projects can be completed within a comparatively short duration, thereby yielding quick results.

High quality data: Leveraging progressive sequencing chemistry methods and image analysis algorithms, the Illumina platform is capable of producing high-quality sequencing data.

Difference Between Illumina Sanger and NGS

The pivotal distinction between Sanger sequencing and NGS lies in the scale of sequencing. While Sanger methodology permits sequencing of only a single DNA fragment at a time, NGS operates on a massively parallel scale, enabling simultaneous sequencing of millions of fragments per run. This process entails sequencing hundreds to thousands of genes concurrently. NGS also confers enhanced discovery potential, facilitating the detection of novel or rare variants through deep sequencing.

Table 1 Differences between Sanger sequencing and NGS

Sanger sequencing is suitable for:

• Investigating diseases with well-defined phenotypes.
• Sequencing one or two genes or up to 96 target loci.
• Performing sequencing runs for 1 to 96 samples without the need for barcode sequences.
• Validating NGS variants with an accuracy rate of up to 99.99%.
• Obtaining longer sequencing read lengths (up to 1000 bp).

NGS is suitable for:

• Studying diseases with higher levels of phenotypic heterogeneity.
• Sequencing more than two genes or more than 96 target loci.
• Sequencing multiple targets within more than 96 samples.
• Discovering novel variants.