Ilumina sequencing

Question 1

What is the most common method of next generation sequencing?

Answer 1

Ilumina sequencing

Question 2

What does PCR occur on in ilumina sequencing?

Answer 2

Fixed points on a glass slide

Question 3

First step of clonal bridge amplification?

Answer 3

The genome is fragmented into tiny pieces

Question 4

What is attached to either end of the DNA fragments in clonal bridge amplification?

Answer 4

Adaptors

Question 5

What is specific about the adaptor that isnt in contact with the slide in clonal bridge amplification?

Answer 5

It has sequence complementarity to the primer

Question 6

Where is the primer in clonal bridge amplification?

Answer 6

On the glass slide, next to the primer and fragment hybrid

Question 7

Why does the adaptor on the top have sequence complementarity to the primer in clonal bridge amplification?

Answer 7

It allows it to bridge over

Question 8

What does each DNA fragment result in in clonal bridge amplification?

Answer 8

One cluster of cloned fragments

Question 9

What is specific about the nucleotides used in reversible tye terminator chemistry?

Answer 9

They are modified

Question 10

How is the base group of the nucleotides in reversible dye terminator chemistry modified

Answer 10

There is a dye attached to the base group, which is cleavable

Question 11

How is the -OH group of the nucleotides modified in RDTC?

Answer 11

It has a terminator group instead of the H

Question 12

What does the terminator group do in RDTC?

Answer 12

Blocks the addition of the next nucleotide

Question 13

WHat is special about the terminator group in RDTC?

Answer 13

It is reversible, meaning it can be changed back to an OH group

Question 14

Why is the terminator group in RDTC reversible?

Answer 14

So it can be changed back so the next nucleotide can be added

Question 15

How many types of nucleotide are added at once in RDTC?

Answer 15

all four

Question 16

What happens to the reaction immidiately after one nucleotide is added?

Answer 16

It terminates

Question 17

Why does the reaction terminate after the addition of one nucleotide in RDTC?

Answer 17

The terminator group prevents more nucleotides from being added

Question 18

What is added to each nucleotide in RDTC?

Answer 18

Each one has a different colour dye added

Question 19

What happens to the dye/fluorophore after its nucleotide has been added?

Answer 19

It is excited, causing it to give off the colour

Question 20

How can you tell which nucleotide has been added?

Answer 20

By observing the colour given off

Question 21

What happens after a colour is given off?

Answer 21

A chemical is added to reverse the terminator group so another nucleotide can be added

Question 22

What is the accuracy of ilumina sequencing?

Answer 22

> 99.9%