Next Generation Sequencing

Question 1

What is PCR used for?

Answer 1

→ To amplify a specific region of DNA
→ So you have sufficient material to sequence for other reactions

Question 2

What does each cycle of PCR achieve?

Answer 2

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

Question 3

What are the disadvantages of Sanger Sequencing?

Answer 3

→ Slow and low throughput

→ Costly

→ Usually for single gene tests

Question 4

What are the 4 core principles in next generation sequencing?

Answer 4

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

Question 5

What does DNA library construction involve?

Answer 5

→ You need to prepare the DNA sample
→ It is chopped into small fragments (300bp) (shearing)

Question 6

What is a DNA library?

Answer 6

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

Question 7

How is shearing done?

Answer 7

→ Chemically
→ Enzymatically
→ Physically (sonication)

Question 8

What is sonication?

Answer 8

Firing sound waves at DNA

Question 9

How are the sheared ends of the DNA repaired?

Answer 9

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

Question 10

What is the end result of shearing?

Answer 10

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

Question 11

What do adapters contain?

Answer 11

→ 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

Question 12

What does a flowcell contain?

Answer 12

DNA library fragments

Question 13

How does hybridization occur?

Answer 13

→ The flowcell is flooded with DNA fragmetns
→ They attach to the surface of the flowcell
→ Random process

Question 14

Why do you need to amplify fragments?

Answer 14

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

Question 15

How are clusters generated?

Answer 15

Bridge amplification

Question 16

Describe how sequencing by synthesis occurs

Answer 16

→ 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

Question 17

What are the requirements for sequencing by synthesis?

Answer 17

→ DNA polymerase
→ Chain terminator
→ Bases with different fluorescent dye colours
→ Sequence each single nucleotide 1 cycle at a time in a controlled manner

Question 18

What happens after sequencing by synthesis?

Answer 18

→ 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

Question 19

What does the sequencing machine give you?

Answer 19

→ Short sequences and a base call
→ It tells you how confident it is that the base is correct

Question 20

What happens during analysis of the sequencing by synthesis?

Answer 20

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

Question 21

How do you look for genetic variants using NGS?

Answer 21

You compare the consensus sequence with the human genome reference

Question 22

What is the difference between NGS and Sanger sequencing?

Answer 22

→ NGS - digital readout
→ Sanger - analogue readout

→ Sanger - one sequence read
→ NGS - consensus sequence of many reads

Question 23

What are the applications of NGS?

Answer 23

Exome sequencing

Question 24

Describe how target enrichment works

Answer 24

→ 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

Question 25

Why is target enrichment done?

Answer 25

We are only interested in 1-2% of the genome

Question 26

How is NGS used in disease identification?

Answer 26

→ Perform exome sequencing
→ Compare variant profiles of affected individuals
→ Find if a disease is heterozygous

Question 27

Describe how RNA Seq works

Answer 27

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.

Question 28

Why is RNA Seq used?

Answer 28

NGS of RNA samples determines which genes are actively expressed

Question 29

What are the advantages of RNA Seq?

Answer 29

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

Question 30

What can RNA Seq be used to discover?

Answer 30

Distinct isoforms of genes that are differentially regulated and expressed

Question 31

What is third generation sequencing?

Answer 31

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

Question 32

What are the advantages of oxford nanopore sequencing?

Answer 32

→ No expensive machine needed
→ Each flow cell is the machine itself
→ Scalable to required throughput

Question 33

What are the disadvantages of nanopore sequencing?

Answer 33

→ Expensive
→ High error rates
→ Developing technology

Question 34

What are P5 and P7 adaptors?

Answer 34

Anchors for attachment of library fragments to the flow cell

Question 35

What can the number of sequencing reads produced from each gene in RNA-Seq be used for?

Answer 35

Used as a measure of gene abundance

Question 36

What are the applications of exome sequencing?

Answer 36

→ Collecting disease affected individuals and their families
→ Gene identification
→ Compare variant profiles of affected individuals
→ Identify the variant or mutation shared by the affected individuals

Question 37

What is involved in exome data analysis?

Answer 37

→ Sequence Read Alignment
→ Target Coverage Reporting
→ Variant calling
→ Variant annotation