during Group 1 Library 4 MinION run I had some issues with low pore occupancy. I was originally concerned that, by increasing the quantity of DNA retained through end-prep, I’d “overloaded” the adapter ligation step, resulting in a high proportion of un-adapted DNA. I thought this unadapted DNA might then be lowering the effective library concentration, resulting in low occupancy.
However, after some discussion in a Nanopore community forum thread with David Eccles, I don’t think adapters are the issue. Instead, David suggested the problem may lay with the short reads “leaving the pores hanging,” and taking too long for a given pore to “find” a new read to sequence. His recommendation was to spike in some longer control DNA. My timeline is too short to order any additional lambda phage DNA, but I can use the DNA Control Sample (DCS) provided in the Native Barcoding Kit (~3.6kb).
With this re-introduction of DCS to the library prep protocol, I need to re-do the Group 2 library.
| Catalog # | Conc. (ng/uL) | uL for 1000ng | uL for 11uL | Barcode |
|---|---|---|---|---|
| 51861 | 94.1 | 10.63 | 0.37 | 18 |
| 51892 | 137 | 7.3 | 3.7 | 19 |
| 51732 | 157 | 6.37 | 4.63 | 20 |
Followed Native Barcoding 96 V14 gDNA protocol, using DCS, with the following modifications:
1000ng input DNA from each sample
retain 3.75uL DNA through end-prep
Qubit 1:
Standard 1: 31.32
Standard 2: 11873.41
G2L2: 17.7 ng/uL
Qubit 2:
Standard 1: 32.99
Standard 2: 14756.63
G2L2: 24.2 ng/uL