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.