Next Generation Sequencing Flashcards

1
Q

What is PCR used for?

A

→ To amplify a specific region of DNA

→ So you have sufficient material to sequence for other reactions

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What does each cycle of PCR achieve?

A

→ Each cycle doubles the amount of DNA copies in the target sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the disadvantages of Sanger Sequencing?

A

→ slow and low throughput

→ Costly

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are the 4 core principles in next generation sequencing?

A

→ 1) DNA library construction
→ 2) Cluster Generation
→ 3) Sequencing by synthesis
→ 4) Data analysis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What does DNA library construction involve?

A

→ You need to prepare the DNA sample

→ It is chopped into small fragments (300bp) (shearing)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is a DNA library?

A

→ A collection of random DNA fragments of a specific sample to be used for further study

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How is shearing done?

A

→ Chemically
→ Enzymatically
→ Physically (sonication)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is sonication?

A

→ Firing sound waves at DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How are the sheared ends of the DNA repaired?

A

→ Adenine nucleotide overhangs are added to the ends of fragments
→ This is done by polymerase
→ and then Thymine overhangs can be ligated to the adenine overhangs on the DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the end result of shearing?

A

→ A DNA library of small random stable fragments representative of the original sample

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What do adapters contain?

A

→ Components to allow the library fragments to be sequenced
→ Sequencing primer binding sites
→ P5 and P7 anchors for attachment of library fragments to the flow cell

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What does a flowcell contain?

A

→ DNA library fragments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How does hybridization occur?

A

→ The flowcell is flooded with DNA fragmetns

→ they attach to the surface of the flowcell

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Why do you need to amplify fragments?

A

→ You cannot measure individual molecules they are too small
→ need to amplify so you can measure them

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How are clusters generated?

A

→ Bridge amplification

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Describe how sequencing by synthesis occurs

A

→ The DNA fragments are attached to the flowcell
→ DNA polymerase adds complementary modified bases with a terminator at the end to the flowcell fragments
→ The bases have different fluorescent dyes attached to them
→ the 4 bases are imaged with a photograph
→ the terminator chemical group is cleaved with an enzyme
→ process is repeated until the flowcell fragment is double stranded

17
Q

What are the requirements for sequencing by synthesis?

A

→ Chain terminator
→ bases with different fluorescent dye colors
→ DNA polymerase

18
Q

What happens after sequencing by synthesis?

A

→ The camera sequentially images all 4 bases on the surface of the flowcell each cycle
→ the cycle image is converted into a nucleotide base call

19
Q

What does the sequencing machine give you?

A

→ Short sequences and a base call

→ it tells you how confident it is that the base is correct

20
Q

What happens during analysis of the sequencing by synthesis?

A

→ the short read sequences need to be re-assembled like a jigsaw
→ a consensus sequence needs to be generated of the original DNA sample

21
Q

How do you look for genetic variants using NGS?

A

→ you compare the consensus sequence with the human genome reference

22
Q

What is the difference between NGS and Sanger sequencing?

A

→ NGS - digital readout
→ Sanger - analogue readout
→ Sanger - one sequence read
→ NGS - consensus sequence of many reads

23
Q

What are the applications of NGS?

A

→ Exome sequencing

24
Q

Describe how target enrichment works

A

→ Incubate RNA that is complementary to the exons
→ DNA library is hybridized with RNA baits
→ Magnetic beads are added (streptavidin)
→ The exon sequences are pulled out
→ RNAse is added to digest the RNA

25
Q

Why is target enrichment done?

A

→ we are only interested in 1-2% of the genome

26
Q

How is NGS used in disease identification?

A

→ perform exome sequencing
→ compare variant profiles of affected individuals
→ find if a disease is heterozygous

27
Q

Describe how RNA Seq works?

A
  1. Extract RNA from cells.
  2. Convert the RNA to cDNA using Reverse Transcriptase before making the DNA library.
  3. NGS of RNA samples determine which genes are actively expressed.
28
Q

Why is RNA Seq used?

A

→ NGS of RNA samples determines which genes are actively expressed

29
Q

What are the advantages of RNA Seq?

A

→ The number of sequencing reads produced from each gene can be used as a measure of abundance
→ quantification of expression levels

30
Q

What can RNA Seq be used to discover?

A

→ distinct isoforms of genes that are differentially regulated and expressed

31
Q

What is third generation sequencing?

A

→ DNA passing through a nanopore and a base sequence is converted to an electrical current

32
Q

What are the advantages of oxford nanopore sequencing?

A

→ No expensive machine needed
→ each flow cell is the machine itself
→ scalable to required throughput

33
Q

What are the disadvantages of nanopore sequencing?

A

→ Expensive
→ high error rates
→ developing technology