TITLE: ONT Q20+ (V12) Adapter Ligation for Fungal DNA Barcoding
AUTHORS: Stephen        Douglas Russell, Stephen   Douglas Russell

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

Tested with:
Flowcells: Flongle 9.4.1 or MinION 10.4.1
Ligation Kit: V12 - LSK112

Time required: ~45 minutes

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

AMX H -  
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 - Lig  

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                                                     9.4.1 Flongle Volume            R10.4.1 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
Adapter Mix H (AMX H)                          2.5 μl                                          5 μl
Total                                                           50 μl                                           100 μ
SECTION: Adapter Ligation
6. 5 s Spin down with a mini centrifuge for 5 s.

SECTION: Adapter Ligation
7. Incubate the reaction for 10 min at room temperature.

SECTION: Adapter Ligation
8. Resuspend magnetic bead stock by vortexing.
SECTION: Adapter Ligation
9. Add 20 µL (Flongle) or 40 µL (R10.4) 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.

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 100-250 μl 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. Just flick 10 times or so.

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 15 μl Elution Buffer (EB). Spin down for 5 s and incubate for 10 min at room temperature. 

SECTION: Adapter Ligation
16. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.
SECTION: Adapter Ligation
17. Remove and retain 15 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 1uL 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), we are looking for 
Flongle R9.4.1: 5 fmol - 20 fmol = .003ug - .012ug of DNA.
MinION R9.4.1: 5 - 50 fmol = .003ug - .029ug of DNA.
MinION R10.4.1: 25 - 75 fmol = .015ug - .044ug of DNA.

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

22ng         1ug
--------   *   --------       =   0.022ug/uL    *   15uL (elution buffer; or 13 if you quantified using 2uL)  =  0.33 ug DNA in sample of 15uL elution buffer
 1uL         1000ng

*Note: the 0.33 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. You could always calculate

Flongle 9.4.1
How much additional molecular water to have 5.5uL needed for the next step give us correct amount of DNA? 
0.33ug / xuL = 0.010ug (17 fmol DNA)   x = 33uL   x 5.5uL  =  181.5uL - 15uL = 166.5uL

So at 0.022ug/uL quantification, add an additional 166.5uL of molecular water to have right concentration to use 5.5uL for the next step with Flongle. 

11ng/uL sample comes out to adding an additional 78.5 uL of molecular water.
31ng/uL sample comes out to adding an additional 221.65 uL of molecular water.

I would use 150 uL if you are not able to quantify your sample.

MinION R9.4.1
0.33ug / xuL = 0.025ug (42 fmol DNA)   x = 13.2uL   x 11uL =  145.2uL - 15uL = 130uL elution buffer addition.

MinION R10.4.1
0.33ug / xuL = 0.04ug (67 fmol DNA)   x = 8.25uL   x 11uL =  90.75uL - 15uL = 75uL elution buffer addition.