Once you mix your 5 minute transposon step, another SPRI step is needed to kill the transposon enzyme and then a motor protein is added. This is unique to ONT sequencing. This pooled library just needs to quantitated on a Tape Station to get the concentration b4 loading.
The Flongles want 3-20 femptomoles of DNA.
They are tiny. 30uls are loaded.
Once you load this DNA, 10,000 reads in 30 minutes and 300,000 overnight.
These are single molecule reads that require GPUs for basecalling.
Validating PCR assays has never been easier. This is why there is no excuse for clinical primers sets used in Covid to go to market without sequence data.
This is a $5K Oxford Nanopore Sequencer.
The flow cell (flongle) is $90 and is a real time sequencer.
In this study we are amplifying with two different ITS primers sets.
One set is 450bp in length and doesn’t amplify the host ITS region (Cannabis).
The other 750bp product captures plants and fungi.
ITS regions are from rDNA and organize the tree of life
These are the key regions used to classify the taxonomy or eukaryotes. Bacteria use 16S regions and viruses have no such conserved regions to organize around.
Amplifying and sequencing these regions is the most common way microbiome studies are performed.