NGS, WGS, WES

Question 1

Explain how PCR may be used to generate many copies of a DNA segment.

Answer 1

• The polymerase chain reaction (PCR) uses a thermocycling reaction to amplify DNA.
• In addition to the DNA sample of interest, the reagents needed are primers, deoxynucleoside triphosphates (dNTPs), a heat-resistant (e.g. taq) polymerase, a buffer solution and bivalent cations - usually Mg²⁺ - to allow the polymerase to work.
• One cycle of PCR typically involves 3 main steps - denaturation, annealing, extension.
• Denaturation: DNA sample is heated to 94-98°C for 20-30 secs, causing denaturation by breaking the hydrogen bonds between the dsDNA.
• Annealing: temp lowered to 50-65°C for 20-40 secs, allowing primers to anneal to the 3’ ends of the sense and antisense strands. The exact temperature must be low enough to allow the primers to bind but high enough to ensure the primers bind specifically to their complementary target sequence.
• Extension: the temperature is increased to ~72°C (close to the optimum temperature for the polymerase). The taq polymerase then adds complementary dNTPs to the template DNA strand, extending the new strand in the 5’ to 3’ direction until the target DNA has been copied to generate identical copies of dsDNA.
• Thus, each cycle of PCR doubles the quantity of target DNA, and so many cycles of PCR generate an exponential increase in the amount of DNA. 20-40 cycles are usually needed to generate sufficient DNA for subsequent applications.

Question 2

A researcher has amplified a 751bp piece of DNA by PCR, and now wants to determine the DNA sequence in several members of a family. Describe in detail an appropriate method that could be used.

Answer 2

• Sanger sequencing is appropriate since this is a relatively small DNA segment.
• Sanger sequencing utilises chain termination to generate many DNA fragments of different sizes, which can then be visualised by capillary electrophoresis.
• The DNA sample of interest is combined with DNA polymerase, primers, the four dNTPs and the four di-deoxynucleoside triphosphates (ddNTPs), each of which is labelled with a different fluorophore (fluorescent dye). The ddNTPs are chain terminating since they lack a 3’ hydroxyl (OH) group to form a phosphodiester bond with the elongating DNA strand.
• As in PCR, the target DNA is first denatured to generate a ssDNA template.
• Next, primer annealing and extension occur.
• The respective ddNTPs are present in a low concentration relative to the dNTPs. Thus, ddNTPs are only occassionally incorporated into the elongating DNA. When this happens, the chain is terminated and a DNA fragment of characteristic size is generated.
• Capillary electrophoresis is then used, where DNA fragments migrate from the cathode to the anode, with smaller DNA molecules moving faster. Thus, the size order of fragments corresponds to the DNA sequence.
• As fragments migrate, they pass through a laser and detector which analyse and record the fluorescence of the tagged ddNTP.
• This process is automated to generate a sequential readout of the fluorescence peaks, which reflect the DNA sequence.

Question 3

Outline the three-step process of next-generation sequencing.

Answer 3

1. DNA library construction
2. Cluster generation
3. Sequencing-by-synthesis

Question 4

Explain how a DNA library is constructed in NGS.

Answer 4

• DNA is chopped into small 300bp fragments - known as shearing.
• This can be achieved chemically, enzymatically or physically.
• The ends of DNA fragments are repaired by adding adenine (A) overhangs.
• Adapters with thymine (T) overhangs are ligated to the DNA fragments.
• Adapters contain primer binding sites and P5 and P7 anchors for attachment of library fragments.
• The end result is a DNA library of billions of small, stable, random fragments representative of our original DNA sample.

Question 6

Explain how cluster generation is carried out in NGS.

Answer 6

• Hybridise DNA library fragments to flowcell.
• Perform bridge PCR on the surface of flowcell, generating clusters.
• Many billions of clusters are generated from a single library fragment.
• Clusters are then big enough to be visualised and are loaded onto the sequencing platform.

Question 7

Explain how sequencing-by-synthesis is carried out in NGS.

Answer 7

• One cycle consists of:
  
  • Single nucleotide incorporation
  • Flowcell wash
  • Imaging the 4 bases
  • Cleavage of terminator group and dye with enzyme
• Camera sequentially images 4 bases on the surface of flowcell for each cycle.
• Each cycle image is converted to a nucleotide base call (ACGT).
• Cycle number anywhere between 50-250bp.
• Short read sequences from the machine are reassembled using a reference genome and bioinformatics software.

Question 8

Explain how whole exome sequencing is carried out.

Answer 8

• Target enrichment
• Capture target regions of interest with complementary RNA baits
• Baits are tagged and a purification step removes unwanted sequences
• Potential to capture 30-60Mb genomic regions

Question 9

Describe the 2 main differences between Sanger and NGS readouts.

Answer 9

• NGS produces a digital readout. Sanger produces an analogue readout.
• Sanger is one sequence read. NGS is a consensus sequence of many reads.

Question 10

How can NGS be used to determine the level of gene expression?

Answer 10

RNA-sequencing:

• RNA first converted to cDNA and then library generated.
• NGS of RNA samples determines which genes are actively expressed.
• Single experiment can capture the expression levels of thousands of genes.
• Number of sequencing reads from each gene indicates gene abundance, which can be quantified to give expression level.
• Differences in expression level of all genes in the experiment can be calculated.
• RNA-seq can be used to discover distinct gene isoforms that are differentially expressed and regulated.

Question 11

Outline the approach of third-generation sequencing.

Answer 11

• Oxford nanopore sequencing.
• Single-molecule sequencing.
• No PCR involved.
• DNA passes through a nanopore and base sequence converted to electrical current.