Illumina sequencing | DNA sequencing by synthesis

Summary

The transcript describes the development and significance of the sequencing-by-synthesis technology, initially developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. This technology, commercialized by Solexa and later acquired by Illumina, has become a leading platform in next-generation sequencing (NGS). The process involves clustering DNA on a flow cell surface, followed by a stepwise addition of fluorescently labeled nucleotides, allowing high-throughput and accurate sequencing. However, challenges include noise and errors from uncleaved fluorophores in later sequencing cycles.

Takeaways

• 🔬 The sequencing-by-synthesis technology used by Illumina was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge.
• 🏢 Solexa, a company founded by Balasubramanian and Klenerman in 1998, commercialized this sequencing method.
• 💼 Illumina acquired Solexa in 2007 and has significantly advanced the original technology.
• 🧬 Illumina's sequencing platform is currently the most widely used in the next-generation sequencing (NGS) field.
• 🧪 Illumina's method involves clustering DNA on a flow-cell surface using 'bridging amplification' to create clusters of identical, single-stranded DNA molecules.
• 📸 After primer annealing, fluorescently labeled nucleotides are added one at a time to the primer, and a camera captures the fluorescence from each cluster.
• 🔗 The 3' hydroxyl protection group and the fluorophore are enzymatically cleaved to continue the sequencing cycle.
• 📊 Illumina's stepwise sequencing is advantageous for sequencing homopolymers, which can be challenging for other platforms.
• 🚀 The throughput of Illumina sequencers is 10-100 times higher per run compared to other sequencing platforms.
• ⚠️ A potential issue with Illumina's technology is the accumulation of uncleaved fluorophores or protection groups, which can increase noise and substitution errors in later cycles.

Q & A

What technology did Illumina acquire to build its sequencing platform?

Illumina acquired Solexa, a company founded by Shankar Balasubramanian and David Klenerman, to commercialize their sequencing-by-synthesis technology.

What is sequencing-by-synthesis technology?

Sequencing-by-synthesis is a technology that involves sequencing DNA by synthesizing the complementary strand and detecting each incorporated nucleotide in real-time.

How does Illumina's sequencing platform cluster DNA?

Illumina's sequencing platform clusters DNA on a flow-cell surface using a method called 'bridging amplification,' which creates clusters of identical single-stranded DNA molecules.

What role do fluorescently labeled nucleotides play in Illumina's sequencing process?

Fluorescently labeled nucleotides are added to the 3' end of the primer based on the complementary base of the template strand. A camera detects the fluorescence to identify the last incorporated nucleotide in each cluster.

How does Illumina prevent the addition of multiple nucleotides per cycle?

Illumina uses chemically protected 3' hydroxyl groups on the fluorescently labeled nucleotides, which prevent the addition of more than one nucleotide per cycle.

What challenge does sequencing homopolymer regions pose, and how does Illumina's technology address it?

Sequencing homopolymer regions, which are repeating stretches of a single type of nucleotide, can be challenging for other sequencing platforms. Illumina's stepwise addition of sequencing reactions provides accuracy in sequencing these regions.

What advantage does Illumina's sequencing platform have in terms of throughput?

Illumina's sequencing platform offers a throughput that is 10-100 times higher per sequencing run compared to other sequencing platforms.

What is paired-end sequencing, and why is it beneficial?

Paired-end sequencing involves reading both ends of a DNA fragment, which compensates for shorter read lengths and increases accuracy by providing two reads of the same DNA template.

What is a potential drawback of Illumina's sequencing technology?

A potential drawback is the accumulation of uncleaved fluorophores or protective groups, which can cause noise and increase substitution errors in later sequencing cycles.

How has Illumina improved the original Solexa sequencing technology?

Illumina has built upon and rapidly expanded the original Solexa technology, making it one of the most widely used platforms in the next-generation sequencing (NGS) field.

Outlines

00:00

🔬 Illumina's Sequencing Technology: Origins and Advancements

This paragraph discusses the development and commercialization of sequencing-by-synthesis technology by Shankar Balasubramanian and David Klenerman at Cambridge University. They founded Solexa in 1998 to bring this technology to market, which was later acquired by Illumina in 2007. Illumina has significantly expanded the original technology, making their sequencers the most widely used in next-generation sequencing (NGS). The paragraph details the process, including clustering DNA on a flow-cell surface through bridging amplification, the use of fluorescently labeled nucleotides, and the stepwise addition of sequencing reactions, which are particularly effective for sequencing homopolymers. It also highlights Illumina's high throughput and paired-end sequencing capabilities, which improve accuracy.

05:28

⚠️ Challenges in Illumina Sequencing: Accumulation of Fluorophores

This paragraph outlines the primary challenge associated with Illumina sequencing technology, which is the potential accumulation of uncleaved fluorophores or protective groups during each sequencing step. This buildup can lead to increased noise and higher substitution errors, especially in the later cycles of sequencing. The paragraph emphasizes how this issue can impact the overall accuracy and reliability of the sequencing results.

Keywords

💡Sequencing-by-synthesis

Sequencing-by-synthesis is a DNA sequencing technology utilized by Illumina. It involves synthesizing a new strand of DNA complementary to the template strand, with the incorporation of fluorescently labeled nucleotides. This method is central to Illumina’s sequencing platforms and allows for high-throughput sequencing by detecting the fluorescence emitted as each nucleotide is added.

💡Solexa

Solexa is a company founded in 1998 by Shankar Balasubramanian and David Klenerman at the University of Cambridge to commercialize their DNA sequencing technology. Illumina acquired Solexa in 2007 and further developed its sequencing technology, making it one of the most widely used platforms in next-generation sequencing (NGS).

💡Illumina

Illumina is a leading biotechnology company that specializes in sequencing and array-based technologies. After acquiring Solexa in 2007, Illumina enhanced and expanded the sequencing-by-synthesis technology, making it the most commonly used platform in the field of next-generation sequencing (NGS). Illumina sequencers are known for their high throughput and accuracy.

💡Next-generation sequencing (NGS)

Next-generation sequencing (NGS) refers to advanced DNA sequencing technologies that allow for massive parallel sequencing, significantly increasing the speed and throughput of sequencing. Illumina’s platforms, based on sequencing-by-synthesis, are a dominant force in NGS, providing more extensive and accurate data than earlier sequencing methods.

💡Bridging amplification

Bridging amplification is a process used in Illumina’s sequencing platforms to cluster DNA on a flow cell surface. This method amplifies single-stranded DNA to create clusters of identical sequences, allowing for efficient and simultaneous sequencing of millions of DNA molecules. It is a key step in the sequencing-by-synthesis process.

💡Fluorescently labeled nucleotides

Fluorescently labeled nucleotides are modified nucleotides used in sequencing-by-synthesis, where each nucleotide (A, T, C, G) is attached to a fluorescent dye. These nucleotides are added to the growing DNA strand during sequencing, and the fluorescence emitted upon their incorporation allows for the detection and identification of each nucleotide added, enabling the determination of the DNA sequence.

💡3' hydroxyl protection group

The 3' hydroxyl protection group is a chemical modification that temporarily blocks the 3' end of a nucleotide during sequencing, preventing the addition of more than one nucleotide per cycle. This step ensures the accurate, stepwise addition of nucleotides, which is critical for reading homopolymer regions and maintaining sequencing accuracy.

💡Homopolymer

A homopolymer is a sequence of DNA consisting of repeated nucleotides of the same type (e.g., AAAA). These sequences can be challenging to sequence accurately, but Illumina’s sequencing-by-synthesis method, with its stepwise nucleotide addition, addresses this issue more effectively than some other sequencing platforms.

💡Paired-end sequencing

Paired-end sequencing is a technique used in next-generation sequencing where both ends of a DNA fragment are sequenced. This approach increases accuracy and allows for better alignment and detection of structural variations in the DNA sequence. Illumina’s platforms commonly use paired-end sequencing to compensate for shorter read lengths.

💡Substitution error

Substitution error refers to a type of sequencing error where one nucleotide is incorrectly identified as another. In Illumina sequencing, this can occur due to the accumulation of uncleaved fluorophores or protection groups, which can introduce noise and errors, particularly in later cycles of sequencing. Managing these errors is crucial for ensuring high accuracy in sequencing results.

Highlights

The sequencing-by-synthesis science was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge.

Solexa was founded in 1998 to commercialize the sequencing method developed by Balasubramanian and Klenerman.

Illumina acquired Solexa in 2007 and has since expanded the technology rapidly.

Illumina sequencers are currently the most widely used platform in the next-generation sequencing (NGS) field.

Illumina uses a flow cell surface for clustering DNA through 'bridging amplification,' which generates clusters with millions of identical, single-stranded DNA molecules.

Fluorescently labeled nucleotides are added to the 3' end of the primer based on the complementary base of the template strand.

A camera takes a snapshot of the flow cell to detect the fluorescence from the last incorporated nucleotide of each cluster.

The 3' hydroxyl protection group and the fluorophore are enzymatically cleaved to proceed to the next cycle of sequencing.

Illumina's stepwise addition of sequencing reactions is beneficial when sequencing homopolymers, which can be challenging for other platforms.

The throughput of Illumina sequencers per run is 10-100 times higher than that of other sequencing platforms.

Illumina's paired-end sequencing capabilities offer enhanced accuracy by reading the same DNA template twice.

A potential issue with Illumina sequencing technology is the accumulation of uncleaved fluorophores or protection groups, which can cause high noise and increased substitution errors in later cycles.

Transcripts

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