TITLE: FUNDIS ONT V14 Nanopore Adapter Ligation for Fungal DNA Barcoding Flongle 10.4.1
AUTHORS: Stephen Douglas Russell, Harte Singer

[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
Ligation Kit: V14 - LSK114

Time required: ~45 minutes

Adapted from dx.doi.org/10.17504/protocols.io.dm6gpb5zdlzp/v5

[STEPS]
SECTION: Adapter Ligation Flongle 10.4.1
1. You should already have your reagents on ice from the dA tailing step, but if not you will need the NEBNext Quick Ligation Module enzyme and from the ONT kit you will need 1 tube of SFB, and LA, LNB, EB, and AXP. Spin down the NEB enzyme and immediately place on ice. Thaw AXP and allow to come up to room temperature, do not place on ice. Thaw other reagents at room temperature, flick-mix, spin down, and keep on ice. 

LNB, LA, AXP, SFB, EB -   
Quick T4 Ligase -  

SECTION: Adapter Ligation Flongle 10.4.1
2. In a 1.5 mL DNA LoBind tube, mix in the following order:

Between each addition, pipette mix 10-20 times.


 

SECTION: Adapter Ligation Flongle 10.4.1
3. Spin down with a mini centrifuge for 2 s.

SECTION: Adapter Ligation Flongle 10.4.1
4. Incubate the reaction for 10 min at room temperature. This is a good time to do a flow cell check and get your computer set up. 

SECTION: Adapter Ligation Flongle 10.4.1
5. Resuspend AMPure XP (AXP) magnetic bead stock by vortexing.
SECTION: Adapter Ligation Flongle 10.4.1
6. Add 20 µL  of resuspended beads to the reaction and mix by flicking the tube.

SECTION: Adapter Ligation Flongle 10.4.1
7. Incubate for 5 min at room temperature, ONT suggests using a Hula mixer, you can just gently flick the tube a few times. 

SECTION: Adapter Ligation Flongle 10.4.1
8. Spin down the sample for 2 s and pellet on a magnet for 2 min. 

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

SECTION: Adapter Ligation Flongle 10.4.1
9. Wash the beads by adding 125 µL  of Short Fragment Buffer (SFB). Flick the beads vigorously to resuspend (they will only partially resuspend), spin down for 2 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.


SFB -    

SECTION: Adapter Ligation Flongle 10.4.1
10. Repeat the previous step.   

SECTION: Adapter Ligation Flongle 10.4.1
11. Spin down for 2 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 Flongle 10.4.1
12. Remove the tube from the magnetic rack and resuspend the pellet in 7 µL Elution Buffer (EB). Incubate for 10 min at room temperature.  Use this time to put away all of the reagents from the previous steps and set up your reagents for the final library mixing step. Use the glass vials that come with the Flongle kit, you will need FCF (Flow Cell Flush) SB (Sequencing Buffer) LIB (Library Beads). You will also need the FLT (Flow Cell Tether) From the main Ligation Sequencing kit. Make sure you have your Qubit reagents ready for quantification. 

SECTION: Adapter Ligation Flongle 10.4.1
13. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 min.

SECTION: Adapter Ligation Flongle 10.4.1
14. 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
15.  Quantify 2 µL of your ligated library using the Qubit fluorimeter 

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. ONT recommends that you dilute the library in Elution Buffer if required, however we use molecular grade water instead. We dilute the entire 5uL of library, however you may dilute a smaller amount if you choose. 

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 = 6ng - 12ng of DNA in 5uL of final dilute library.

For a 22 ng/uL sample

22ng/uL    *   5uL  =  110ng DNA in sample of 5uL elution buffer. 

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

C1V1 = C2V2, rearrange C1V1/C2 = V2

(110ng * 5uL) / 10ng(17 fmol DNA) = 55uL total volume

Bring initial volume (5uL) up to 55uL = add 50uL molecular water

So at 22ng/uL quantification, add an additional 50uL of molecular water to have right concentration to use 5uL for the next step with Flongle.