Next Generation Sequencing

Question 1

Briefly describe the Polymerase Chain Reaction

Answer 1

• Fundamental principle for any DNA sequencing application
• Used to amplify a specific region of DNA, Primers flank the region you want to amplify
• Each cycle doubles the amount of DNA copies of your target sequence
• Amplify enough DNA molecules so that we have sufficient material

Question 2

Briefly describe Sanger sequencing

Answer 2

• Invented by Fred Sanger in 1977
• Cycle sequencing
• Based on PCR (also needs a PCR product as an input)
• Needs primers
• Modified nucleotides: Chain terminators or nucleotide specific colour tag

Question 3

What does Sanger sequencing identify?

Answer 3

• A single nucleotide polymorphism (SNPs) or mutations
• Can identify monogenic disease causing mutations
• Often used for single gene tests

Question 4

What are the benefits of next generation DNA sequencing?

Answer 4

• Decrease in the cost of DNA sequencing
• Since the end of 2007, the cost has dropped at a faster rate than that of Moore’s law

Question 5

When was Next generation of DNA sequencing developed?

Answer 5

• Development of NGS methods began 13 years ago with 454 pyrosequencing
• DNA sequencing throughput jumped 10 orders of magnitude
• Solexa sequencing by synthesis (SBS) developed end of 2005

Question 6

What happened as a result of the development of next generation sequencing?

Answer 6

• Replaced Sanger sequencing for almost all sequencing in the lab
• Whole genome sequencing
• Whole Exome sequencing

Question 7

What are the four steps of NGS?

Answer 7

1. DNA library construction
2. Cluster generation
3. Sequencing by synthesis
4. Data analysis

Question 8

Describe the steps in DNA library construction (PART 1)

Answer 8

• Takes place in the wet lab
• DNA is first chopped into small fragments (typically 300 bp). called shearing
• Can be achieved chemically, enzymatically or physically (sonication)

Question 9

Describe the steps in DNA library construction (PART 2)

Answer 9

• The end of the sheared DNA fragments have to be repaired
• Adenine nucleotide overhangs are added to end of fragments
• Adapters with Thymine overhangs can be ligated to the DNA fragments

Question 10

Describe the steps in DNA library construction (PART 3)

Answer 10

• Adapters contain the special 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 11

Describe the steps in cluster generation (PART 1)

Answer 11

• Hybridise DNA library fragments to flowcell
• But we can’t visualise individual single molecules of our DNA library: too small
• We need to amplify the fragments to a bigger size for a stronger signal

Question 12

Describe the steps in cluster generation (PART 2)

Answer 12

• Perform bridge amplification to generate clusters
• Many billions of clusters originating from single DNA library molecules
• Clusters are now big enough to be visualised
• Flow cell is now ready to be loaded

Question 13

Describe the steps in Sequencing By Synthesis (PART 1)

Answer 13

Modified 4 bases (ATCG) with:
- Chain terminators
- Different fluorescent colour dye

• Sequence each single nucleotide 1 cycle at a time in a controlled manner

Question 14

Describe the steps in Sequencing By Synthesis (PART 2)

Answer 14

• Single Nucleotide incorporation (DNA polymerase)
• Flowcell wash
• Image the 4 bases (digital photograph)
• Cleave terminator chemical group and dye with enzyme

Question 15

Describe the steps in Sequencing By Synthesis (PART 3)

Answer 15

• Camera sequentially images all 4 bases on the surface of the flowcell each cycle
• Each cycle image is converted to a nucleotide base cell
• Cycle number is anywhere between 50 - 600 nucleotide base pairs

Question 16

Describe the last stage: Analysis of NGS data

Answer 16

• Short read sequences from the sequencing machine need to be pieced together like a jigsaw
• Mapping locations of our sequence reads on the reference genome sequence
• To generate a consensus sequence of original DNA sample library

Question 17

Compare and contrast between NGS and Sanger Sequencing

Answer 17

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

Question 18

How many genes are there in the human genome?

Answer 18

21,000 Genes

Question 19

What part of the gene are we usually interested in?

Answer 19

In the gene protein coding exons or ‘Exome’ which represents 1-2% of the genome

Question 20

What percentage does pathogenic mutations acquaint for?

Answer 20

80% of pathogenic mutations are protein coding

Question 21

What can we do with the whole exome sequencing to be more efficient?

Answer 21

More efficient to only sequence the bits we are interested in rather than the whole Exome sequencing

Question 22

What is Whole Exome Sequencing used for?

Answer 22

• Target enrichment
• Capture target regions of interest with baits
• Potential to capture several Mb genomic regions of interest
• Exome would be 50 Mb in size

Question 23

How is Exome Data Analysis carried out?

Answer 23

Sequence read alignment -> Target Coverage Reporting -> ? -> Variant Annotation

Question 24

How is Whole Exome Sequencing used?

Answer 24

• The target is to look for protein coding mutations in the exon
• The patient DNA sample is subjected to Exome sequencing
• This should show a snippet of the consensus sequence of the sequence sample
• this reveals a heterozygous mutation in the CFTR gene

Question 25

What are the applications of Exome Sequencing?

Answer 25

- Collecting disease affected individuals and their families - Use of NGS in disease gene identification - Perform Exome sequencing - Compare variant profiles of affected individuals

Question 26

What is RNA sequencing 1?

Answer 26

RNA-seq experiments use the total RNA (or mRNA) from a collection of cells or tissue

Question 27

What occurs in RNA-sequencing?

Answer 27

- RNA is converted to cDNA before library construction - NGS of RNA samples are used to determine which gene are actively expressed - Single experiment can capture the expression levels of thousands of genes

Question 28

What occurs in RNA-sequencing? (PART 2)

Answer 28

- The number of sequencing reads produced from each gene can be used as a measure of gene abundance - Quantification of the expression levels - Calculation of the differences in gene expression of all genes in the experimental conditions