After my preliminary Flongle runs (Group 1 Library 1 and Group 1 Library 2), I’m a little worried about how much MinION output I’ll get, so I’m going to switch to 3 samples per flow cell.
I’ve also looked at some options for library prep adjustments:
Exclude FFPE Repair. FFPE repair, in part, repairs cytosine deamination C->U damage. Since I;m interested in potentially examingin patterns of age-related deamination, I’d like to exclude this step if possible. It may cause problems, though, since the un-repaired DNA damgae (e.g. nicks) may affect sequencing, by e.g. clogging the pores more quickly.
Begin with 1000ng gDNA dor each sample, instead of 400ng, when multiplexing <= 4 samples.
| Catalog # | Conc. (ng/uL) | uL for 1000ng | Label/Barcode |
|---|---|---|---|
| 1606826 | 408 | 2.5 | 09 |
| 1740336 | 368 | 2.7 | 10 |
| 1740363 | 343 | 2.9 | 11 |
End-Prep Master Mix (no FFPE):
| 1X | 4X | |
|---|---|---|
| Ultra II Buffer | 1.75uL | 7uL |
| Ultra II Mix | 0.75uL | 3uL |
Made 4X and added 2.5uL of Master Mix to each tube
Qubit 1 (barcodes ligated, in 35uL)
Standard 1: 37.40
Standard 2: 9926.48
Library 3: 22.8 ng/uL
Qubit 2 (adapters ligated, in 15uL)
Standard 1: 31.61
Standard 2: 11333.17
Library 3: 26.2 ng/uL