What is Illumina Sequencing?
Illumina sequencing is a second-generation sequencing technology that has become a powerful tool for genomics research. The method uses reversible dye terminator technology to detect the sequence of a DNA molecule. The process begins by fragmenting the DNA sample into short fragments, typically 100-150 base pairs (bp) in length. These fragments are then ligated to a universal adapter and annealed to a slide. Polymerase chain reaction (PCR) amplification is performed to create a large number of copies of each fragment, resulting in clusters on the slide.
Sequencing slides contain fluorescently labeled nucleotides, DNA polymerase, and terminators. During sequencing, one base is added at a time and the fluorescent signal from the incorporated nucleotide is detected. The terminator is then removed, allowing the next base in the sequence to be added. Through a series of cycles, the computer decodes the fluorescent signal and reconstructs the DNA sequence in a relatively short period of time (typically 4 to 56 hours).
Advantages of Illumina Sequencing
- High accuracy: Illumina sequencing is 99% accurate, making it ideal for applications requiring precise sequence determination. This accuracy is critical in other areas where clinical diagnosis and reliability are essential.
- High throughput: The technology is capable of generating large numbers of reads in parallel, enabling high-throughput sequencing. Illumina instruments are capable of generating billions of short reads in a single run, resulting in comprehensive coverage of the genome or transcriptome.
- Proven infrastructure: Illumina has built a robust ecosystem around its sequencing platform, including a broad range of compatible library preparation kits, analysis software, and a large user community. This infrastructure provides researchers with reliable tools and resources to support their sequencing experiments.
What is Nanopore Sequencing?
Nanopore sequencing represents a third-generation sequencing technology that uses protein nanopores to detect the nucleic acid sequence of DNA or RNA molecules. Oxford Nanopore Technologies, a leading company in this field, manufactures nanopore sequencing devices that use this innovative approach.
In nanopore sequencing, a flow cell with tiny nanopores embedded in a resistive membrane is used. Each nanopore corresponds to its own electrode, connected to the channel and sensor chip. When a DNA or RNA molecule passes through a nanopore, its current is modulated. The change in current is influenced by the shape, size and length of the DNA or RNA sequence, resulting in a characteristic wave pattern. The wavy lines are then decoded in real time to identify specific DNA or RNA sequences. Explore with our Nanopore Direct RNA Sequencing Analysis for more information.
Advantages of Nanopore Sequencing
- Long read lengths: One of the key advantages of nanopore sequencing is its ability to generate long read lengths, which can extend into the hundreds of kilobases range. This feature is particularly valuable for applications such as ab initio genome assembly, complex region analysis, and structural variation detection.
- Real-time analysis: Unlike other sequencing technologies, nanopore sequencing enables real-time analysis because the waveform pattern is decoded as the DNA or RNA molecule passes through the nanopore. This feature allows immediate insight into sequence composition, making nanopore sequencing a promising tool for remote rapid diagnosis and in situ analysis.
- Portability: The compact size and portability of nanopore sequencing devices make them attractive for a variety of field applications. Researchers can perform sequencing experiments in resource-limited environments or perform real-time monitoring of environmental samples, viral outbreaks, or microbial populations.
Similarities Between Illumina and Nanopore Sequencing Technologies
While the underlying principles of Illumina and Nanopore sequencing technologies are different, they also exhibit certain similarities:
- DNA and RNA sequencing: Both Illumina and Nanopore sequencing can be used to sequence DNA and RNA molecules. They provide versatile platforms for studying genomic, transcriptomic and epigenomic information.
- Fast sequencing: Both technologies offer fast sequencing capabilities, enabling researchers to obtain sequencing results in a relatively short period of time compared to traditional Sanger sequencing.
- High Accuracy: Both Illumina and Nanopore sequencing offer high levels of accuracy, although Illumina sequencing is typically slightly more accurate.
Differences Between Illumina and Nanopore Sequencing Technologies
While Illumina and Nanopore sequencing are both powerful sequencing methods, there are significant differences between the two:
- Sequencing method: Illumina sequencing utilizes a reversible dye terminator technique in which one fluorescently labeled nucleotide is doped at a time. In contrast, nanopore sequencing uses protein nanopores to detect changes in the current of a DNA or RNA molecule as it passes through the nanopore.
- Read Length: Illumina sequencing produces shorter read lengths, typically up to 500 base pairs (bp), while nanopore sequencing excels at producing long read lengths, extending into the hundreds of kilobases range.
- Accuracy: Illumina sequencing is accurate up to ~99%, while nanopore sequencing is typically 92-97% accurate.
How to Choose Between Illumina and Nanopore Sequencing Technologies
In the realm of DNA sequencing, the decision to opt for Illumina or Nanopore sequencing technologies warrants careful consideration, taking into account a multitude of factors that can significantly impact the outcome of a research project. Delving into the intricacies of this choice requires a deep understanding of the complexities and specificities associated with each technology.
One pivotal aspect to weigh when selecting a sequencing technology is the alignment of project goals with the capabilities of each platform. The unique research objectives and applications at hand hold significant sway in this decision-making process. Should the pursuit involve long read lengths and real-time analysis, the allure of nanopore sequencing becomes evident. Conversely, if the research demands a higher degree of precision or necessitates compatibility with an already established infrastructure, the scales may tip in favor of Illumina sequencing, which offers a well-established ecosystem to cater to these requirements.
Another fundamental consideration is the nature of the sample itself. The quality, quantity, and complexity of the DNA or RNA sample under scrutiny exert profound influences on the suitability of a sequencing technology. In instances where the genome exhibits a high level of complexity or the sample harbors regions that pose challenges, nanopore sequencing emerges as a viable option due to its long read lengths, which can help unravel intricate genomic architectures more effectively.
However, the decision-making process is incomplete without a meticulous evaluation of the budgetary implications associated with each technology. Researchers must take into account not only the upfront costs of instrument acquisition but also the ongoing expenses encompassing consumables and data analysis. In this regard, the cost-effectiveness of Illumina sequencing, coupled with its well-established ecosystem, may present a compelling proposition for projects operating within specific financial constraints.
Conclusion
In culmination, it becomes apparent that Illumina and Nanopore sequencing technologies each possess distinct strengths and capabilities within the domain of DNA sequencing. Illumina's forte lies in its exceptional accuracy, high throughput, and robust infrastructure. Conversely, nanopore sequencing offers the advantage of long read lengths, real-time analysis, and portability. Consequently, researchers embarking on the selection process between these two powerful sequencing technologies must exercise due diligence by thoroughly assessing their project requirements. Crucial factors such as read length, accuracy, sample characteristics, and budgetary considerations should be meticulously scrutinized to ensure an informed decision aligning the chosen technology with the research objectives at hand.
References
- Wang Y, Zhao Y, Bollas A, et al. Nanopore sequencing technology, bioinformatics and applications[J]. Nature biotechnology, 2021, 39(11): 1348-1365.
- Kircher M, Heyn P, Kelso J. Addressing challenges in the production and analysis of illumina sequencing data[J]. BMC genomics, 2011, 12: 1-14.
