Pyrosequencing + Illumina NGS

In pyrosequencing, what is released during the incorporation of each deoxynucleotide that is essential for generating the sequencing signal?

• Chemiluminescent dye

• ATP

• Pyrophosphate (PPi)

• Deoxynucleoside triphosphate (dNTP)

What enzyme in pyrosequencing is responsible for converting pyrophosphate (PPi) into a detectable chemiluminescent signal?

• DNA polymerase

• Luciferase

• Sulfurylase

• Nucleotidase

During the pyrosequencing process, why are deoxynucleotides added sequentially rather than all at once?

• To avoid continuous light flashes and ensure accurate sequence detection

• To ensure complete incorporation of each nucleotide before moving to the next

• To prevent the degradation of the DNA template

• To reduce the risk of contamination

What is the role of emulsion PCR in massively parallel pyrosequencing?

• To degrade unincorporated nucleotides before the next one is added

• To convert pyrophosphate (PPi) into a chemiluminescent signal

• To denature double-stranded DNA into single-stranded templates

• To amplify DNA fragments in an aqueous droplet, each containing a single DNA fragment attached to a bead

In pyrosequencing, how are DNA fragments prepared for attachment to small metallic beads?

• By directly attaching the fragments to the beads without any modification

• By denaturing the fragments into single strands and binding them to the beads with a primer

• By ligating adaptors that facilitate binding through biotin-streptavidin linkages

• By performing PCR amplification directly on the beads

In Illumina NGS, what is the purpose of attaching adaptors to DNA fragments during library preparation?

• To prevent DNA degradation during sequencing

• To enable the DNA fragments to bind to the sequencing flow cell and allow amplification

• To fluorescently label the DNA fragments for detection

• To increase the length of the DNA fragments for better sequencing accuracy

What is the primary function of "bridge amplification" in Illumina NGS?

• To sequence the DNA fragments by synthesizing complementary strands

• To add fluorescently labeled nucleotides to the DNA fragments

• To cleave the terminator group from nucleotides after each incorporation

• To create clusters of identical DNA sequences from a single DNA molecule

During the sequencing by synthesis (SBS) step in Illumina NGS, what allows the identification of the newly added nucleotide?

• The sequence of the DNA fragment

• The length of the DNA fragment

• The color of the fluorescent signal emitted by the nucleotide

• The number of clusters on the flow cell

Why are nucleotides used in Illumina sequencing referred to as "reversible terminators"?

• They block the addition of further nucleotides, but this block can be removed

• They are permanently bound to the DNA strand once incorporated

• They fluoresce multiple times during sequencing

• They prevent DNA polymerase from functioning after incorporation

What happens to the short sequences (reads) generated from each cluster during the final stages of Illumina NGS?

• They are discarded if they do not match the reference genome

• They are aligned to a reference genome or assembled to identify genetic variants

• They are sequenced again to ensure accuracy

• They are stored in a database without further analysis