Next Generation Sequencing Flashcards
What is PCR used for?
→ To amplify a specific region of DNA
→ So you have sufficient material to sequence for other reactions
What does each cycle of PCR achieve?
→ Each cycle doubles the amount of DNA copies in the target sequence
What are the disadvantages of Sanger Sequencing?
→ Slow and low throughput
→ Costly
→ Usually for single gene tests
What are the 4 core principles in next generation sequencing?
1) DNA library construction
2) Cluster Generation
3) Sequencing by synthesis
4) Data analysis
What does DNA library construction involve?
→ You need to prepare the DNA sample
→ It is chopped into small fragments (300bp) (shearing)
What is a DNA library?
A collection of random DNA fragments of a specific sample to be used for further study
How is shearing done?
→ Chemically
→ Enzymatically
→ Physically (sonication)
What is sonication?
Firing sound waves at DNA
How are the sheared ends of the DNA repaired?
→ 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
What is the end result of shearing?
A DNA library of small random stable fragments representative of the original sample
What do adapters contain?
→ 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
What does a flowcell contain?
DNA library fragments
How does hybridization occur?
→ The flowcell is flooded with DNA fragmetns
→ They attach to the surface of the flowcell
→ Random process
Why do you need to amplify fragments?
→ You cannot measure individual molecules they are too small
→ Need to amplify so you can measure them
How are clusters generated?
Bridge amplification
Describe how sequencing by synthesis occurs
→ 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
What are the requirements for sequencing by synthesis?
→ DNA polymerase
→ Chain terminator
→ Bases with different fluorescent dye colours
→ Sequence each single nucleotide 1 cycle at a time in a controlled manner
What happens after sequencing by synthesis?
→ 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
What does the sequencing machine give you?
→ Short sequences and a base call
→ It tells you how confident it is that the base is correct
What happens during analysis of the sequencing by synthesis?
→ The short read sequences need to be re-assembled like a jigsaw
→ A consensus sequence needs to be generated of the original DNA sample
How do you look for genetic variants using NGS?
You compare the consensus sequence with the human genome reference
What is the difference between NGS and Sanger sequencing?
→ NGS - digital readout
→ Sanger - analogue readout
→ Sanger - one sequence read
→ NGS - consensus sequence of many reads
What are the applications of NGS?
Exome sequencing
Describe how target enrichment works
→ 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
