Question

Illumina Sequencing short answer questions

A. Suppose that there was a mistake and the nucleotides you ordered for sequencing were all labeled with blue tags. How would this affect the results? Explain your reasoning.

B. When template is added to the flow cell for bridge PCR, the concentration of template is critical. What would happen if too many template molecules were added to the flow cell?

Answer 1

A. Illumina sequencing is a technology for sequencing DNA molecule. In this method single stranded DNA molecule are combined with adapter molecule and then transfer to the surface containing flow cells that will bind with adapter molecule of single stranded DNA. Amplification of this single strand DNA is done and denature again and anchored to the substrate molecule. Ultimately Million of Double stranded DNA is generated in each channel of flow cell. Four labelled terminal nucleotides are used for stop the DNA synthesis then signal production by laser beam. 4 different colour of signal produce for each nucleotide in the output. Then this signal will be aligned and compare with the references.

If by mistake only blue colour labelled nucleotide is ordered then 4 different nucleotides are not identified from one another, all the signal produced in the output shows blue signal. This one signal colour in the output will confuse the researcher to sequence the DNA molecule.

B. If too many single stranded DNA molecules are added to the flow cells then random binding of adapter molecule of template with in the flow cell will occur, and it will produce noise in the final signal. Concentration of template should be critical for illumine sequencing, higher template concentration will cause improper amplification in bridge PCR and that will cause an improper sequencing in the flow cell.