How Does Illumina Sequencing Work

Illumina sequencing is a next-generation sequencing method, which is also called the “sequencing-by-synthesis” method. Illumina sequencing is involved in processing millions of fragments in parallel. The four basic steps involved in the Illumina sequencing workflow are library preparation, cluster generation, sequencing, and data analysis, which are further described in this article.

Key Areas Covered

1. What is Illumina Sequencing
     – Definition, Facts, Advantages
2. How Does Illumina Sequencing Work
     – Process of Illumina Sequencing:
          – Library Preparation
          – Cluster Generation
          – Sequencing
          – Data Analysis

Key Terms: Cluster Generation, Data Analysis, Illumina Sequencing, Library Preparation, Sequencing by Synthesis

What is Illumina Sequencing

Illumina Sequencing or sequencing-by-synthesis (SBS) technology is the most widely-used next-generation sequencing technology in the world. More than 90% of the sequencing data of the world are generated by Illumina sequencing. It was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. They founded a company known as Solexa in 1998. Then Illumina purchased Solexa in 2007, rapidly improving the original technology. Hence, the method is also called Solexa/Illumina sequencing method. The main advantage of Illumina sequencing is that it delivers a high yield of error-free reads.

How Does Illumina Sequencing Work

The four steps involved in Illumina sequencing are described below.

Step 1. Library Preparation

• A sequencing library is prepared by simultaneous tagmentation of DNA into short segments of 200-600 base pairs by transposases in a process known as tagmentation, followed by ligation of adaptor into both 3′ and 5′ ends of the short segments of DNA.
• Additional motifs such as sequencing primer binding site, index, and a region, which is complementary to flow cell oligo are added to the adaptor on both sides by reduced cycle amplification. Tagmentation and addition of motifs are shown in figure 1.

Figure 1: Tagmentation and Addition of Motifs

Step 2. Cluster Generation

• The prepared sequencing library is denatured and loaded into a flow cell for cluster generation. During cluster generation, each fragment in the sequencing library is isothermally amplified. The flow cell is made up of glass containing lanes. Each lane is coated with two types of oligonucleotides. One type is complementary to the 5′ region of the additional motifs and the other type is complementary to the 3′ region of the additional motifs of the prepared library. Hence, these oligos bind to the corresponding regions of DNA in the sequencing library. The flow cell with two types of oligos is shown in figure 2. The oligo that binds to the 5′ region of the sequencing library is pink in color while the oligo that binds to the 3′ region of the sequencing library is green in color.

Figure 2: Flow Cell

• Once the single-stranded sequencing library is bound to the oligo, the complementary strand is generated by DNA polymerase. Then, the resultant double-stranded DNA is denatured and the original strand is washed away.
• The clonal amplification of the fragment is achieved through bridge amplification. During this process, the strand folds over the second type of oligo on the flow cell. Then, polymerase synthesizes the double-stranded bridge. The denaturation of the bridge results in two DNA strands: both forward and reverse strand on the oligos of the flow cell.
• Bridge amplification is repeated over and over to obtain simultaneously millions of clusters of all types of fragments in the sequencing library by clonal amplification. Clonal amplification is shown in figure 3.

Figure 3: Clonal Amplification

• Then the reverse strands are washed away, retaining only the forward strands on the flow cell. In the forward strand, the 3′ end is free and it is blocked in order to prevent unwanted priming.

Step 3. Sequencing

First Read of the Reverse Sequence

• Sequencing begins with the extension of the first sequencing primer. Illumina sequencing method uses modified dNTPs, which contain a terminator at the 3′ position of the deoxyribose sugar. These dNTPs are also fluorescently-labeled in different colors.

• After the addition of each complementary nucleotide, the clusters in the flow cell are observed for the emission of fluorescence.

• After the detection of light, the fluorophore can be washed off.

• Then the terminator group of the 3′ position of the sugar is regenerated by a hydroxyl group, allowing the addition of a second dNTP to the growing chain. This process is known as sequencing-by-synthesis. The sequencing-by-synthesis is shown in figure 4.

Figure 4: Sequencing-by-Synthesis

• At the completion of the synthesis, the first read of the reverse sequence is obtained and the sequencing product is washed away.

Index 1 Read

• The index 1 primer is then hybridized to clusters to generate a second read in the same way by sequencing-by-synthesis. The sequencing product is washed away.

Index 2 Read

• The 3′ end of the cluster is then deprotected, allowing the hybridization of the 3′ end with the second type of oligo on the flow cell (green color). By this, the sequence of the index 2 region is obtained. The sequencing product is washed away.

Second Read of the Forward Sequence

• The second type of oligo is extended by a polymerase, forming a double-stranded bridge. The bridge is denatured and their 3′ ends are blocked. The forward strand is washed away.
• The second read of the forward sequence is obtained through sequencing-by-synthesis by the hybridization and extension of the second sequencing primer.

Step 4. Data Analysis

• The billions of reads obtained by sequencing are grouped based on their index sequences.
• Then, the sequences with similar reads are clustered.
• Forward and reverse reads are paired to form contiguous sequences.
• The ambiguous alignments can be resolved by paired sequences.
• The contiguous sequences are aligned to the reference genome for variant identification.

The following video explains the complete process of Illumina sequencing.

Conclusion

Illumina sequencing is a next-generation sequencing method. Illumina sequencing is involved in the preparation of a sequencing library with 200-600 base pairs long fragments of DNA. The four steps involved in the Illumina sequencing are library preparation, cluster generation, sequencing, and data analysis. Since Illumina sequencing gives sequence reads with high accuracy, it is the widely-used sequencing method in the world.

Reference:

1. “Sequencing by Synthesis (SBS) Technology.” Sequencing Technology | Sequencing by Synthesis, Available here.

Image Courtesy:

1. “DNA Processing Preparation” By DMLapato – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Oligonucleotide chains in Flow Cell” By DMLapato – Own work, (CC BY-SA 4.0) via Commons Wikimedia
3. “Sequencing by synthesis Reversible terminators” By Abizar Lakdawalla (talk) – I created this work entirely by myself (CC BY-SA 3.0) via Commons Wikimedia
4. “Cluster Generation” By DMLapato – Own work (CC BY-SA 4.0) via Commons Wikimedia