Performing library prep on extracted, cleaned, and QC’d DNA for Nanopore sequencing.
Setup
As a reminder, we’ll be multiplexing 4 samples per MinION flow cell (E. tourneforti genome is 600Mb, so 10X is 6Gb; anticipate 30Gb MinION output; so 5 samples max)
I also want to group samples for multiplexing by DNA integrity and quantity, since the protocol includes sleecting a pooled library quantity based on average fragment lengths.
Using the TapeStation peak fragment size as a proxy for average size (reasonable, due to normal distributions of fragment lengths), I selected 4 samples with ihghest fragment size for Group 1:
| Catalog # | peak bp | tube label |
|---|---|---|
| 1606826 | 1906 | 1 |
| 1740336 | 1698 | 2 |
| 1740363 | 1696 | 3 |
| 1740407 | 1798 | 4 |
Following the Oxoford Nanopore Tecnologies Ligation Sequencing gDNA - Native Barcoding 96 V14 (SQK-NBD114.96) protocol (document version NBE_9171_v114_revS_02Jul2025).
Using Short Fragment Buffer (SFB), not LFB. I don’t have any LoBind PCR tubes, so will be using 0.2mL 8X strip tubes for steps before barcodes are pooled.
Notes
Native Barcoding 96 Kit
REF: SQK-NBD114.96
LOT: NBD1496.40.0007
Flongle Expansion Kit
REF: EXP-FSE002
LOT: FSE002.30.0005
| Catalog # | Extraction (ng/uL) | uL for 400ng | Barcode # |
|---|---|---|---|
| 1606826 | 408 | 1 uL | 01 |
| 1740336 | 368 | 1.1 uL | 02 |
| 1740363 | 343 | 1.2 uL | 03 |
| 1740407 | 260 | 1.5 uL | 04 |
Before beginning library prep:
ensure there is an available thermal cycler with the following program: 20C for 5min, 65C for 5min
Pre-heat an incubator plate to 37C
Qubit 1 (Barcodes ligated, in 35uL)
Standard 1: 39.06
Standard 2: 16073.91
Library 1: 1.62 ng/uL
Qubit 2 (Adapters ligated, in 15uL)
Standard 1: 30.11
Standard 2: 14133.11
Library 1: 2.56 ng/uL
Library has very low concentration. This is likely because, while I started with 400ng input DNA from each sample, only a fraction of this is carried through from end-prep to barcode ligation. At Step 7 of Barcode Ligation, for each sample, 0.75uL of end-prepped DNA is used, added to 3uL of nuclease-free H2O. The protocol is likely optimized for multiplexing a large number of samples at once, but I’m just using 4.
I think I should be able to, instead, carry-over a full 3.75uL of end-prepped DNA at this step, and exclude additional nuclease-free water. I’m going to try another round of library prep using the same samples, making this change to retain more DNA.
I’ll call the first round of these samples “Library 1”, and the second round with more DNA carried over “Library 2.”
Library 2
Same samples (“Group 1”), but need to assign new unique barcodes.
| Catalog # | Extraction (ng/uL) | uL for 400ng | Barcode # |
|---|---|---|---|
| 1606826 | 408 | 1 uL | 05 |
| 1740336 | 368 | 1.1 uL | 06 |
| 1740363 | 343 | 1.2 uL | 07 |
| 1740407 | 260 | 1.5 uL | 08 |
During Step 7 of Barcode Ligation, excluded nuclease-free H2o and used 3.75uL end-prepped DNA (instead of the recommended 0.75uL).
Qubit 1 (Barcodes ligated, in 35uL)
Standard 1: 33.38
Standard 2: 13411.37
Library 2: 13.7 ng/uL
Qubit 2 (Adapters ligated, in 15uL)
Standard 1: 30.11
Standard 2: 14133.11
Library 2: 16.5 ng/uL
Yay! Library 2 has much more DNA in the final library. I’ll need to run both Library 1 and Library 2 on Flongles to ensure the higher DNA retention doesn’t also carry over inhibiting reagents from end-prep that might affect sequencing.
Took both Library 1 and Library 2 thorugh Step 18 of Adapter Ligation & Clean Up. Did not dilute to Final Library, since I’ll need different concentrations for Flongle and MinION flow cells.