Jordans Notes 11 Flashcards
What substitution model takes into account
different rates of transversion vs transition
Kimura 2 parameter model
What two methods can be used to determine which
substitution model is best?
- Akaike Information Criterion
- Bayesian Information Criterion
What are the three applications of next generation
sequencing?
- identifying multiple species
- SNPs genotyping
- Investigating the entire genome rather than a select few areas
What is the first stage of second generation
sequencing?
constructing a DNA library
What are the three steps of library prep
- Fragmentation
- Adapter ligation
- PCR amplification
What happens after fragmentation but before
ligation during SGS
The ends are repaired and an A tail is added
to allow the attachment of adapters with a corresponding T tail
How does Illumina sequence the DNA
4 fluorescently labeled nucleotides on a flowcell in parallel
Nucleotides fluoresce when they are bound to the synthesizing DNA
When the nucleotide binds, its terminator prevents further binding and then a laser causes that nucleotide to fluoresce. Then the fluorescent tag is removed and the inactive 3’ hydroxyl group is activated to allow further synthesis.
How does Ion Torrent sequence DNA
Different nucleotides are flushed in succession over the well containing the DNA.
When one of the four nucleotide flushes successfully results in a binding base pair, it releases a hydrogen ion which is detected by the sensing layer (pH change).
What 5 things are different about 3rd gen
sequencing?
- sequenced in real time
- Single molecules (no PCR)
- Long read lengths
- Cheaper to run
- Currently low accuracy
How does MinIon read the DNA sequence?
Measures ionic charge difference of each basepair as the single stranded DNA pass through the protein nanopore
NO Sequencing by synthesis!!
