Question

Which of the following is true regarding Illumina sequencing? Select all that apply.

The fluorescent label remains on the DNA strand as additional nucleotides are added.

Each of the nucleotides has a different colored label.

The blocking group is removable.

A blocking group is present on the 2' OH of the nucleotide.

Answer 1

• Illumina or solexa sequencing technique is based on sequencing by synthesis.
• The steps include: library preparation, cluster generation and reversible terminator-based sequencing.
• The reverse termination is achieved by 3’-OH blocking efficiency (3’blockers), after primer extension termination.
• The process involves:

1. Immobilization of the sequencing templates and the primers.

2. Primer extension by a base (each with different colored fluorescent tag) and termination.

3. Washing the unincorporated nucleotide and identifying the color of the fluorophore carried by the base of the incorporated nucleotide.

4. Removal of fluorescent label (Fluorescent label removed before adding more nucleotides) and 3’ blocking groups (blockers are removable) from the nucleotides.

5. Washing and repeating steps 2-4.

Answer:

• Each of the nucleotides has a different colored label.
• The blocking group is removable.