How to sequence using Illumina technology

Illumina dye sequencing is a molecular technique for determining the base pair sequence in DNA.

It was developed by Cambridge University’s Shankar Balasubramanian and David Klenerman, who later founded Solexa, which was acquired by Illumina. This method of sequencing is based on a technique known as “bridge amplification,” in which DNA molecules of approximately 500 bp are used as substrates for repeated amplification synthesis reactions on a solid support (i.e., the flow cell) containing oligonucleotide sequences complementary to a ligated adapter. The oligonucleotides on the surface are arranged in such a way that after several rounds of replication, the DNA forms clonal “clusters” of around 1000 copies of each oligonucleotide fragment. Millions of parallel cluster reactions can be accommodated on the flow cell. During the synthesis procedure, patented modified nucleotides corresponding to each of the four bases are added and then detected using a different fluorescent label. The nucleotides also function as reaction terminators, which are unblocked after detection to enable the next round of synthesis. This sequence of reactions is then repeated 300 times or more.

The sequencing workflow consists of four major steps (sample preparation, cluster formation, sequencing, and data analysis), which can be further subdivided as follows (Figures 1 and 2):

1) The first step is to fragment the DNA into more manageable 200-600 base pair segments.

2) Short DNA sequences known as adaptors are ligated to the DNA fragments. The adaptor-ligated DNA fragments are converted into single-stranded forms using sodium hydroxide.

3) The DNA libraries are loaded onto the flow cell. The adaptor DNA binds to complementary sequences on the flow cell’s surface, and unbound DNA is washed away.

4) The DNA bound to the flow cell is then replicated to generate small clusters of DNA.

5) When these molecules are sequenced, they emit a signal powerful enough to be detected by a camera.

6) Unlabeled nucleotides and DNA polymerase are then introduced to the flow cell to extend and link the DNA strands. This results in the formation of ‘bridges’ of double-stranded DNA between the primers on the flow cell surface.

7) Using heat, the double-stranded DNA is broken down into single-stranded DNA, resulting in many million dense clusters of identical DNA sequences.

8) Primers and fluorescently labelled terminators (a type of nucleotide base that stops DNA synthesis) are added to the flow cell.

9) The primer binds to the DNA that is being sequenced.

10) After binding to the primer, the DNA polymerase adds the first fluorescently-labelled terminator to the newly synthesised DNA strand. Once a base is added to the strand of DNA, no additional bases can be added until the terminator base is removed. Lasers are used to activate the fluorescent label on the nucleotide base of the flow cell. A camera detects the fluorescence and records it on a computer. Each terminator base (A, C, G, and T) emits a distinct fluorescent signal.

11) The fluorescently-labelled terminator group is then removed from the first base, and the second fluorescently-labelled terminator base is inserted next to it. This cycle is repeated until millions of clusters have been sequenced.

12) Base-by-base analysis of the DNA fragment occurs during sequencing, making it very accurate. The resulting sequence can then be compared to a reference sequence to look for matches or differences in the sequenced DNA.

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Figure 1 – Descriptive illustration of Illumina sequencing protocol steps 1-6. Source: Illumina Inc.

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Figure 2 – Descriptive illustration of Illumina sequencing protocol steps 7-12. Source: Illumina Inc.

Various methodologies are supported by Illumina sequencing, including whole-genome, exome, and targeted sequencing, metagenomics; RNA-seq; CHIP-seq; and methylome approaches. Throughput varies for each sequencer model, including the MiniSeq, MiSeq, NextSeq, HiSeq, and NovaSeq lines. The MiniSeq can store 7.5 Gb of data and generate 25 million reads per run in segments of 2 x 150bp reads. The MiSeq has a capacity of 15 Gb and can generate 2 x 300bp reads. The NextSeq can provide 120Gb with 400 million reads at a read length of 2 x 150bp. Details about each instrument and its capabilities in relation to specific sequencing projects may be found on Illumina’s website.

Let us know in the comments if you ever had the opportunity to use Illumina technology. How easy or difficult is Illumina sequencing for you?