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
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
→ and then 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
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?
→ Chain terminator
→ bases with different fluorescent dye colors
→ DNA polymerase
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
Why is target enrichment done?
→ we are only interested in 1-2% of the genome
How is NGS used in disease identification?
→ perform exome sequencing
→ compare variant profiles of affected individuals
→ find if a disease is heterozygous
Describe how RNA Seq works?
- Extract RNA from cells.
- Convert the RNA to cDNA using Reverse Transcriptase before making the DNA library.
- NGS of RNA samples determine which genes are actively expressed.
Why is RNA Seq used?
→ NGS of RNA samples determines which genes are actively expressed
What are the advantages of RNA Seq?
→ The number of sequencing reads produced from each gene can be used as a measure of abundance
→ quantification of expression levels
What can RNA Seq be used to discover?
→ distinct isoforms of genes that are differentially regulated and expressed
What is third generation sequencing?
→ DNA passing through a nanopore and a base sequence is converted to an electrical current
What are the advantages of oxford nanopore sequencing?
→ No expensive machine needed
→ each flow cell is the machine itself
→ scalable to required throughput
What are the disadvantages of nanopore sequencing?
→ Expensive
→ high error rates
→ developing technology
