TITLE: ONT V14 Nanopore Adapter Ligation for Fungal DNA Barcoding
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
This process will take your A-tailed library and add the nanopore adapters. Simply combine several chemicals for a single reaction and do a bead cleanup.

Tested with:
Flowcells: Flongle 10.4.1 or MinION 10.4.1
Ligation Kit: V14 - LSK114

Time required: ~45 minutes

[STEPS]
SECTION: Adapter Ligation
1. Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.

LA -   
Quick T4 Ligase -  

SECTION: Adapter Ligation
2. Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing. 

LNB -   

SECTION: Adapter Ligation
3. Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down and place on ice.

EB -   

SECTION: Adapter Ligation
4. Thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.

SFB -   

SECTION: Adapter Ligation
5. In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:

Between each addition, pipette mix 10-20 times.

Reagent                                                     10.4.1 Flongle Volume            10.4.1 MinION Volume
DNA sample from the previous step      30 μl                                           60 μl
Ligation Buffer (LNB)                              12.5 μl                                         25 μl
NEBNext Quick T4 DNA Ligase              5 μl                                              10 μl
Ligation Adapter (LA)                              2.5 μl                                          5 μl
Total                                                          50 μl                                           100 μ
SECTION: Adapter Ligation
6. Spin down with a mini centrifuge for 5 s.

SECTION: Adapter Ligation
7. Incubate the reaction for 10 min at room temperature. Pull the AmPure XP (AXP) from the freezer.

SECTION: Adapter Ligation
8. Resuspend AMPure XP (AXP) magnetic bead stock by vortexing.
SECTION: Adapter Ligation
9. Add 20 µL (Flongle) or 40 µL (MinION) of resuspended beads to the reaction and mix by flicking the tube.

SECTION: Adapter Ligation
10. Incubate on a Hula mixer (rotator mixer) for 5 min at room temperature (or just place in a tube rack without the mixer). Pull the EB and SFB from the freezer.

SECTION: Adapter Ligation
11. Spin down the sample for 5 s and pellet on a magnet for 2 min. 

Keep the tube on the magnet, and pipette off the supernatant.

SECTION: Adapter Ligation
12. Wash the beads by adding 125 µL (Flongle) or 250 µL (MinION) of Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down for 5 s, then return the tube to the magnetic rack for 2 min and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Note: flicking the tube does not seem to fully resuspend the beads. You can choose to flick 10 times or so and spin down. I typically never flick it.

SFB -    

SECTION: Adapter Ligation
13. Repeat the previous step.   

SECTION: Adapter Ligation
14. Spin down for 5 s and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking. 

SECTION: Adapter Ligation
15. Remove the tube from the magnetic rack and resuspend the pellet in 7 µL Elution Buffer (EB). Incubate for 10 min at room temperature.  Start setting up your flowcell on the computer. Hardware and flowcell check. Also pull the Flongle Sequencing Expansion chemicals for loading from the freezer.

SECTION: Adapter Ligation
16. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 min.

SECTION: Adapter Ligation
17. Remove and retain 7 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

Store on ice until you are ready to load in your flowcell.
SECTION: Quantification
18. If you have access to a Quantus or Qubit fluorimeter, now is a good time to quantify 2uL of DNA in your sample. 

It is recommend loading 5 fmol to 10 fmol of this final prepared library onto your flow cells. Loading more than 20 fmol of DNA can reduce the rate of duplex read capture. Dilute the library in Elution Buffer if required.

https://www.promega.com/resources/tools/biomath/

For 900bp length DNA (what our ITS1F-4 rxns appear to average, with adapters), we are looking for:
10 fmol - 20 fmol = .006ug - .012ug of DNA.


For a 22 ng/uL sample (Quantus quantification):

22ng              1ug
--------   *   --------       =     0.022ug/uL             (22/1000=0.022)
1uL                1000ng

0.022ug/uL    *   5uL (elution buffer; or x7 if you did not quantify using 2uL)

=  0.11 ug DNA in sample of 5uL elution buffer. 

*Note: the 0.11 in the calculations below will change based on your individual DNA amount.

**Also note: The ONT protocol suggests using additional EB in order to make concentration adjustments. As there is not a lot of the reagent in the standard packets, and it is not possible to buy more individually, I have been using molecular water for this step with no ill effect. 

Flongle 10.4.1
How much additional molecular water to have 5uL needed for the next step give us correct amount of DNA? 

0.11ug / xuL = 0.010ug (17 fmol DNA)   

x = 11uL   * 5uL  =  55uL - 5uL (or 7 if you did not quanitfy) = 50uL

Overall summary:  ([DNA Concentration] / 1000 * 5 * 100 * 5) - 5 = [Amount of H2O to add]

So at 0.022ug/uL quantification, add an additional 50uL of molecular water to have right concentration to use 5uL for the next step with Flongle. 

11ng/uL sample comes out to adding an additional 22.5 uL of molecular water.
31ng/uL sample comes out to adding an additional 72.5 uL of molecular water.

I would use 50 uL of extra molecular water if you are not able to quantify your sample.