Summary from last 3 library preps and tweaks:
Beginning with and retaining more DNA through prep doesn’t appear to have a negative effect on sequencing (e.g. similar yield from G1 L1 and G1 L2), but it does result in a library with much higher DNA content. This is very important for being able to load the MinION flow cell multiple times (with washes), so for all future library preps I will:
begin with 1000ng DNA input from each sample (or as much DNA as I have, if <1000ng is available)
following end-prep, retain 3.75uL end-prepped DNA during Native Barcode Ligation Step 7, instead of the recommended 0.75uL
Note that, to get 1000ng DNa in 12uL input, will need a sample concentration of >83.3 ng/uL. If input DNA is too dilute, will need to re-concentrate usng Zymo
I realized I don’t use the DNA Control Sample (DCS), since its purpose is to inform, in the event of a failed sequencing run, whether the cause was failed library prep. I’m running libraries on Flongles to confirm successful library prep (without risking a MinION flow cell), so I don’t need the DCS. Instead, this volume can be used for input DNA. For all future library preps I will:
- Exclude the 1uL DCS from each sample prep during DNA Repair and End-Prep Step 6, Instead, makeup the 1000ng input DNA of each sample in 12uL.
Based on the Flongles’ rate of pore degradation and sequencing, I think MinION flow cells may yield less output than I originally estimated. As such, I will only multiplex 3 samples per flow cell.
Excluding FFPE end repair does appear to affect sequencing (G1 L2 vs G1 L3). This reagent will be included in all future library preps.