Run, Group 4 Library, 1 on a minION flow cell for sequencing.
I have a lot of DNA in this library, so I want to try loading 300 fmol DNA to try to increase pore occupancy more (it’s been chronically, low-ish, ~75%).
So I want 300 fmol DNA in 12 uL EB:
G4L1 is 59.2 ng/mL = 157.8 fmol/uL, so let’s do 2uL G4L1 + 10 uL EB.
Flow cell
[ID] FBD08455
1652 pores on 08/14/25 check
1645 pores on 09/05/25 check
Trying the MK1D again after the MinKnow software update:
Hardware check passed
MinION, flow cell health check completed
When setting up sequencing run, turned off pore reserve function, to front load data aquisition.
After update, there are more base modified basecalling options. Selected all options (6mA, 5mC, & 5hmC in all contexts)
Run sequencing on un-primed, un-loaded Flow cell for 4 min to evaluate pore health.
really weird run stats… only 36 pores available on pore scan, almost everything shows “no pore”…
Paused to prime + load.
Okay, after priming + loading, resumed run: Initiated pore scan - 1430 pores
Seeing some “wild flowering” in the channel states panel early on
Occupancy is looking good in first ~15min! ✛ e.g. 47% sequencing, 4.8% available, so (47 / 47 + 4.8) = 90.1%! That’s my highest proportion yet of available pores that are actively sequencing
There’s a notably higher level of adapter than in previous runs - ~7%, compared to previous 1%-2% in MinION runs. This might be expected though, since library is shorter than previous libraries, which means the proportion of adapter:read will be higher
Wash #1
@ 4.5hr in, after ~ ⅔ pore loss (510 pores @ the 3.5hr scan). Very fast pore loss, mostly to the “unavailable” category. Re-loaded w/ 300 fmol again. 783 pores after wash #1
Wash #2
@ 19.5hr in, 84 pores remaining. Re-loaded with 300 fmol, and there are 381 pores after wash/reload.
Wash #3
@ 2 days 1hr in, 40 pores remaining. Re-loaded with 300 fmol. Run errored out when I tried to resume, so I had to restart the MinKnow software and start a new run. 185 pores after run started.