Illumina sequencing is one of the most widely used next-generation sequencing (NGS) technology that uses sequencing by synthesis to detect individual DNA bases as they are added to a growing strand.
It uses advanced instruments capable of processing millions of DNA sequences at the same time. It was originally developed by Solexa and later acquired by Illumina in 2007. The high-throughput nature of Illumina sequencing makes it faster, more accurate, and more cost-efficient than older sequencing techniques such as Sanger sequencing.
It has a similar principle to Sanger sequencing. Both methods use fluorescently-labelled nucleotides and identifies each nucleotide by its fluorescent tag. However, while Sanger sequencing handles one DNA fragment at a time, Illumina is massively parallel and allows millions of fragments to be sequenced simultaneously in a single run.
Illumina offers a range of platforms for various research needs, from benchtop to production-scale applications. The benchtop sequencers, such as the MiSeq and MiniSeq systems, are compact and flexible. They are suitable for labs with smaller sequencing needs. Production-scale sequencers like the NextSeq and NovaSeq systems can be used for large-scale applications.
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Principle of Illumina Sequencing
Illumina sequencing works on the principle of sequencing by synthesis (SBS). This involves identifying DNA bases as they are added to a growing DNA strand. Fluorescently-labeled reversible terminator nucleotides are used and the fluorescence emitted from each added nucleotide is captured. Each of the four DNA bases is labelled with a unique fluorescent dye, allowing the sequencing system to detect which nucleotide has been added during each cycle. The system captures images of these signals which are then used to determine the exact sequence of the DNA fragment.
Illumina sequencing also uses bridge amplification. In this process, DNA molecules with ligated adapters are used as templates for repeated amplification on a solid surface like a glass slide. The slide is coated with oligonucleotides complementary to the adapters, which allows the DNA to form clusters of each fragment. This amplification process creates millions of these clusters on the slide. The clusters enhance the signal and make it easier to detect and differentiate between DNA bases by color.
During sequencing, nucleotides with a unique fluorescent label are added, incorporated, and detected in real time. These nucleotides also act as temporary terminators of DNA synthesis, ensuring that only one base is added at a time which reduces errors and provides high accuracy in reading the sequence. Once imaged, the terminator is cleaved and the next base is added. This cycle is repeated until the entire DNA fragment has been sequenced.
Steps/Process of Illumina Sequencing
1. Nucleic Acid Extraction
The first step in Illumina sequencing is isolating the genetic material from samples of interest. The extraction process is important because the quality of the nucleic acids extracted will directly affect the sequencing results. After extraction, a quality control check is usually performed to ensure the nucleic acids are pure and accurately quantified. UV spectrophotometry is typically used to check the purity, while fluorometric methods are preferred for measuring nucleic acid concentration.
2. Library Preparation
After nucleic acids are isolated, they are prepared for sequencing by creating a library which is a collection of adapter-ligated DNA fragments that can be read by the sequencer. The process starts with DNA fragmentation, where the sample is broken into smaller fragments using methods like mechanical shearing, enzymatic digestion, or transposon-based fragmentation. These fragments undergo end repair and A-tailing to prepare for the attachment of short specific DNA sequences called adapters to both ends of the fragments. These adapters contain sequences that help bind the DNA to the sequencing flow cell. They also include barcode sequences that allow multiple samples to be sequenced simultaneously and distinguished later in the analysis.
3. Cluster Generation by Bridge Amplification
The DNA library is loaded onto a flow cell containing small lanes where amplification and sequencing occurs. The DNA fragments bind to complementary primers attached to the solid surface of the flow cell and undergo bridge amplification. In bridge PCR, each DNA strand bends over to form a bridge on a chip. Forward and reverse primers on the chip help the DNA form these bridges. Each bridge is amplified, creating many clusters at each spot. The process of cluster generation finishes when each DNA spot on the chip has enough copies to produce a strong, clear signal.
4. Sequencing by Synthesis (SBS)
Once clusters are generated, the SBS process begins. Fluorescently labeled nucleotides are added one by one to the growing DNA strand and each nucleotide emits a fluorescence as it attaches. The specific color emitted allows the system to identify the nucleotide. The sequence of each DNA fragment is determined over multiple cycles.
5. Data Analysis
Once the sequencing is completed, the sequences obtained are processed and analyzed using bioinformatics tools. Images collected from each cycle are converted into base sequences by analyzing the fluorescent signals. Bioinformatics tools clean up and organize the data, ensuring the sequences are ready for analysis. Then, the data are analyzed, aligning the sequences to a reference genome or assembling them if a reference is unavailable. This process helps identify sequence variants, map gene locations, and allow downstream analyses. Finally, the data is interpreted to analyze pathways, identify potential biomarkers, or predict gene functions. This step helps translate raw sequencing data into meaningful biological insights. Some Illumina instruments have built-in, easy-to-use analysis software that can help researchers without bioinformatics expertise.
Advantages of Illumina Sequencing
- Illumina sequencing allows millions of DNA fragments to be sequenced in parallel, generating vast amounts of sequencing data in a single run. This makes it ideal for large-scale projects like whole-genome sequencing or transcriptome analysis. This high-throughput approach saves time and resources.
- This method is highly accurate and ensures that sequencing errors are minimal leading to highly reliable results.
- Illumina sequencing allows for rapid sequencing which is particularly useful in clinical settings.
- Illumina sequencing offers a more affordable option for large-scale projects as it is cost-effective compared to traditional sequencing methods like Sanger sequencing.
- This method of sequencing supports both single-read and paired-end libraries.
- This platform supports different sample types and library preparation methods. It can be applied to different samples from whole genomes to targeted regions of interest.
- It is useful for different applications and different research needs.
Limitations of Illumina Sequencing
- Illumina sequencers generate short reads making assembling complex genomes or highly repetitive regions challenging.
- The cost per base is low for this sequencing method but the initial investment in sequencers and maintenance can be expensive for small labs.
- The large volume of data produced requires powerful computational tools and expertise.
- The loss of synchrony during sequencing is also a challenge with Illumina technology. Not all molecules in a cluster incorporate nucleotides at the same time which leads to inaccuracies in the final sequence.
- Another issue is overclustering the support where too much DNA is loaded onto the slide, reducing sequencing quality and accuracy.
Applications of Illumina Sequencing
- Illumina sequencing supports a wide range of applications, including genomic sequencing, RNA sequencing, metagenomics, and ChIP-seq.
- It also supports large-scale genome projects, targeted resequencing, and epigenetic studies, allowing researchers to explore genetic modifications and their implications in disease.
- Sequencing techniques like amplicon sequencing can be used to study microbial communities in microbiome studies. It can be used to study microbial genomes which is important for tracking outbreaks and understanding antibiotic resistance.
- It allows the study of mutations in cancer cells, tracks the progression of the disease, and identifies potential therapeutic targets.
- Illumina sequencing can be used in environmental studies to study biodiversity by sequencing DNA from mixed environmental samples.
- It can be used in forensic science to study the genetic material from crime scenes. This helps to identify suspects or determine relationships between individuals.
References
- NGS Data Analysis for Illumina Platform—Overview and Workflow | Thermo Fisher Scientific – US. (n.d.). Retrieved from http://commerce.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/next-generation-sequencing/ngs-data-analysis-illumina.html
- NGS Workflow Steps | Illumina sequencing workflow. (n.d.). Retrieved from https://www.illumina.com/science/technology/next-generation-sequencing/beginners/ngs-workflow.html
- Principle and Workflow of Illumina Next-generation Sequencing | CD Genomics blog. (2018, October 17). Retrieved from https://www.cd-genomics.com/blog/principle-and-workflow-of-illumina-next-generation-sequencing/
- Principles of Illumina Next-generation Sequencing (NGS) – CD Genomics. (n.d.). Retrieved from https://www.cd-genomics.com/resource-principles-illumina-next-generation-sequencing.html
- Sequencing Platforms | Illumina NGS platforms. (n.d.). Retrieved from https://www.illumina.com/systems/sequencing-platforms.html
- Sequencing Technology | Sequencing by synthesis. (n.d.). Retrieved from https://emea.illumina.com/science/technology/next-generation-sequencing/sequencing-technology.html
- Slatko, B. E., Gardner, A. F., & Ausubel, F. M. (2018). Overview of Next-Generation Sequencing Technologies. Current protocols in molecular biology, 122(1), e59. https://doi.org/10.1002/cpmb.59