Next generation sequencing

Question 1

How many basepairs long is the human genome project?

Answer 1

3 billion basepairs long

Question 2

What technique was used to sequence the human genome project?

Answer 2

All done with the traditional sanger sequencing

Question 3

Cost to complete the human genome project

Answer 3

3 billion dollars

Question 4

What is PCR the fundamental of?

Answer 4

Fundamental for any DNA application

Question 5

What is PCR used for?

Answer 5

Used to amplify a specific region of DNA using primers which flank the region you want

Question 6

What is sanger sequencing and what is it based on?

Answer 6

Cycle sequencing; and its based on the same principles as PCR

Question 7

How are the nucleotides modified in sager sequencing?

Answer 7

Each of the 4 DNA nucleotides have a different dye

Question 8

Disadvantages of sanger sequencing

Answer 8

Costly and slow

Question 9

What is the four step processes in Next generation sequencing?

Answer 9

1. DNA library construction
2. Cluster gene generation
3. Sequencing by synthesis
4. Analysis of NGS data

Question 10

Details of each step in NGS

Answer 10

Refer to notes as it requires understanding

Question 11

NGS vs Sanger sequencing

Answer 11

• NGS produces a digital read out whereas sanger sequencing produces an analogue readout
• Sanger is one sequence read whereas NGS is a consensus sequence of many reads

Question 12

What is NGS also used to study apart from DNA?

Answer 12

Also used to study RNA

Question 13

What does RNA seq use?

Answer 13

RNA seq uses total RNA from a collection of cells and tissues

Question 14

What is done to RNA prior to library construction?

Answer 14

RNA first converted to cDNA

Question 15

What do the NGS of RNA samples determine?

Answer 15

Determine which genes are actively expressed

Question 16

In NGS of RNA, what can a single experiment capture?

Answer 16

Can capture expression levels of a thousand genes

Question 17

In the NGS of RNA, how can we measure gene abundance?

Answer 17

The number of sequence reads produced from each gene can be used as a measure of gene abundance

Question 18

What can we discover in the NGS of RNA with appropriate analysis?

Answer 18

We can discover distinct isoforms of genes

Question 19

Example of third generation sequencing and details

Answer 19

Oxford nanopore sequencing:

• Single molecule sequencing
• No PCR involved
• DNA passes through a nanopore and base sequence is converted into an electrical current