Nanopore sequencing Flashcards
What are the 4 key limitations of all DNA sequencing technologies
- No technology is 100% accurate
- There is a practical limit to the length of DNA that can be sequenced
- Cost
- All sequencing technologies require a molecular handle that the technology can use to grab the DNA
Describe a nanopore flowcell?
A typical flowcell is an array of 512 chambers over which a DNA sample flows.
Each pore is bisected by a synthetic lipid membrane that is punctuated with a protein pore, salt solutions are placed on either side of the membrane and a voltage is applied.
How are individual bases recognised in nanopore sequencing?
Each base has a characteristic size and shape and will block the nanopore channel to a characteristic extent depending on which base it is
What is the name of the enzyme used to feed DNA through a nanopore sequencer?
DNA translocase
How many bases can the DNA translocase enzyme feed through a nanopore sequencer every second?
70-400 bases per second
Describe how DNA from a genome is prepared for nanopore sequencing
- DNA is broken into fragments as a result of processing
- The ends of the input DNA are linked to synthetic DNA molecules called adaptors
- Nanopore adaptors are linked to lipid groups which allow them to diffuse within the lipid membrane and find a nanopore
- When DNA binds to a nanopore, DNA translocase binds and begins feeding the DNA through the pore.
Outline two limitations of Nanopore sequencing
- Nanopores can only practically sequence DNA molecules in the 10,000-50.000 range. The typical length is 33.3Kbp
- There is a limit to accuracy. Nanopore sequencing is only 40-99% accurate and finds it hard to distinguish between homopolymer runs like AAAAAAA VS AAAAAAAAAAA
How much sequence coverage is standard
A) 15X
B) 30X
C) 40X
B) 30X
How many reads per run can you get in a nanopore sequencer?
100,000
Cost per million base pairs for nanopore
£0.35
Run time for nanopore
1.5 days
