NGS preparation

Similar to the First Generation Sequencing technique, in the Illumina sequencing process based on sequencing by synthesis, the nucleotides are individually labeled with a fluorescent dye.

• One fluorescent nucleotide is added, complementary to the DNA sequence to identify, and the elongation stops due to a blocker on its 3' terminal.
• A picture is taken to register the dye/nucleotide.
• The block sitting at the 3' terminal that stopped the elongation is chemically removed from the DNA, allowing the next nucleotide addition to proceed.

The difference with First Generation Sequencing is that parallel sequencing produces millions of reads per run, making it a much more efficient method.

To perform a NGS experiment based on sequencing by synthesis you need:

• Modified DNA sample with adaptors.
• Modified nucleotides.
• T4 polymerase.
• Next Generation Sequencing machine.
• Perform cluster generation.