Presentation 2: MinION Flashcards
What is Karyotyping?
Testing for aneuploidy and CNVs
But, costly, time consuming, and does not sequencing
What is NGS?
Sequence short DNA fragments
Costly and time consuming, large throughput, detect mosaicism
What is 3rd Gen Sequencing?
Sequence nucleotides at the molecular level
- -> Lower cost
- -> Longer fragments
- -> Sequences in parallel and in real-time
- -> Much faster than most NGS
What is MinION?
3rd generation sequencer
What does the MinION Nanopre Sequencer do?
Detects the change in electric currents as single nucleotides on ssDNA pass through a nanowire
Fastest growing 3rd- generation sequencing product
Generates reads in real-time and sequences in parallel
How does the MinION Nanopore Sequencer work?
1) An enzyme unzips the DNA helix into two strands
2) A protein creates a nanopore in the membrane which holds the adapter molecule
3) A flow of ions creates an electric current through the nanowire
4) The adaptor molecule keeps DNA bases in place long enough to be identified electronically
What is the standard MinION Protocol?
Designed to read long fragments for de novo genome assembly
1) Fragment, end repair
2) dA-tail
3) Ligate
4) Sequence
What happens in MinION during ligation?
The DNA forms a loop which can be thread through with help from the hairpin adaptor
Why was the MinION standard protocol altered?
To detect aneuplodies
How was the MinION standard protocol altered?
1) Multiple purifications
2) Reduce volume
3) 4 * C incubation period
4) RT incubation period
How was End Repair and dA tailing developed?
Went from 2 step process to one step process
2 step: 63% ligations in 1+ hour
1 step: 30% ligations in 12 minutes
Then went from one sep process to a one step ultra-fast process, which could do 80% of the ligations in 12 minutes by changing incubation and reducing the reaction volumes
What is and how did the 1 step barcode and adapter ligation help with this research?
Combines barcode ligation and adaptor ligation into one efficient step for ultra fast 1D sequencing.
The adaptor is a Y adaptor, that has a tether attached to it, which helps the DNA to be dragged through the Nanopore much faster
Same accuracy as 2D, and higher throughput in shorter time
What were the results from the Step Ligation?
There is max ligation product when 1D and EM are combined
The TA ligation: DNA fragment + Barcode
6 bp ligation: Barcode + Adaptor
What were the results from the aneuploidy detection?
All aneuplodies detected that were detected in previous 2D studies
What was determined in the pilot test?
Confirmed to be identical to NGS results
Detection of CNVs and mosaics took more time, but accurate
