13. NEXT GENERATION SEQUENCING

Question 1

What is Next-Generation sequencing?

Answer 1

• Next generation sequencing is a broad term used to describe many modern techniques.
• NGS sequences DNA & RNA much quicker than Sanger sequencing & is quicker
• Solexa sequencing by synthesis currently dominates the market

Question 2

What are the four steps of NGS sequencing?

Answer 2

1. DNA library construction
2. Cluster generation (hybridisation & amplification)
3. Sequencing by synthesis
4. Data analysis

Question 3

What is a DNA library?

Answer 3

• A DNA library is a random collection of DNA fragments from a specific sample that will be used for further study

Question 4

How is the DNA library prepared in the lab prior to NGS?

Answer 4

• A DNA sample can be taken from the patient’s blood or from a plant etc.
• The DNA library needs to be chopped into smaller fragments of about 300bp in a process called SHEARING
• Shearing can be CHEMICAL (chemically break down DNA), ENZYMATIC (enzymes digest DNA) or PHYSICALLY (sonification by sound waves)

Question 5

What are the three alterations to the sheared DNA samples in NGS?

Answer 5

• Shearing is a damaging process so the DNA fragments need to be repaired
  1. END REPAIR - The ends of the fragments need to be repaired. An enzyme can convert the single base overhangs into double stranded overhangs
  2. A-TAILING - Adenine overhangs are added to the end of the sequence so that both ends have a poly A tail
  3. ADAPTER LIGATION - An adapter with thymine overhangs can be ligated to the DNA fragment. The adapter contains essential components for the sequencing of the DN fragments

Question 6

What essential components does the adapter have for sequencing of DNA?

Answer 6

1. A sequencing primer binding site

2. P5 & P7 anchor for attachments

Question 7

What is a flow cell?

Answer 7

• A flow cell is a glass slide with lanes containing oligo nucleotides

Question 8

Describe hybridisation in the cluster generation step of NGS?

Answer 8

• The DNA library fragments are placed onto the flow cell & they hybridise with the oligonucleotides
• Hybridisation to the flow cell is a random process but it depends on whether the adapter is complementary to the oligonucleotides
• The DNA fragments are too small to be measured so they need to be amplified
• The double stranded DNA is denatured & the template is washed away before amplification

Question 9

**Describe amplification in the cluster generation step of NGS?

Answer 9

• The DNA fragments are amplified by BRIDGE AMPLIFICATION
• It produces clusters of millions of clonal copies of the original fragments
• Once the clusters are large enough to be visualised, the flow cells can be loaded onto a sequencing machine

Question 10

What two components do the chemically modified bases that will be added contain?

Answer 10

1. Fluorescent base

2. Reversible terminator

Question 11

*What are the steps for sequencing by synthesis?

Answer 11

1. A single base is incorporated by DNA polymerase. The fluorescence of the base can be used to indicate which base it is
2. The flow cell is washed to identify any unbound nucleotides or reagents
3. All 4 bases can be imaged using a camera to see which colour corresponds to which base
4. The chemical group of the reversible terminator is cleaved
   - Normally the terminator stops the incorporation of a new base, but once it’s cleaved a new base can be added
   - These steps are repeated until a full length sequence is achieved

Question 12

How can the NSG data be analysed?

Answer 12

• NSG produces many short reads. These short read sequences need to be re-assembled
• The NSG short reads can be alligned against a reference by mapping out the locations of our sequences
• Once this is done, a consensus of our origina DNA library is generated

Question 13

What’s the difference in the read outs produced by Sanger & NGS?

Answer 13

• Sanger sequencing produces analogue readouts & ONE sequence read
• Sanger sequencing produces a DIGITAL readout & a CONSENSUS of many short reads

Question 14

What is RNA sequencing?

Answer 14

• RNA sequencing uses NGS to identify the presence & quantity of RNA in a sample
• It can determine what genes are actively expressed.
• RNA is converted to cDNA first by reverse transcriptase

Question 15

What are the advantages of whole exome sequencing compared to whole genome sequencing?

Answer 15

• Whole exome sequencing is cheaper than whole genome sequencing
• Whole exome sequencing is more efficient as it only focuses on the coding part of the genome. 80% of pathogenic mutations are located in the coding region