Mar 17, 2023
Version 5
ONT V14 Nanopore Adapter Ligation for Fungal DNA Barcoding V.5
- 1The Hoosier Mushroom Society
Protocol Citation: Stephen Douglas Russell 2023. ONT V14 Nanopore Adapter Ligation for Fungal DNA Barcoding. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpb5zdlzp/v5Version created by Stephen Douglas Russell
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 16, 2023
Last Modified: March 17, 2023
Protocol Integer ID: 78874
Keywords: nanopore, fungi, flongle, fmol, library preparation
Abstract
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
Materials
Reagents
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114 : $694.43 per 6 reactions ($115.74 per MinION run; $57.87 per Flongle run)
NEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S : $361.00 per 20 reactions ($18.05 per MinION run; $9.03 per Flongle run)
*note: This kit has two components. We use one - NEBNext Quick T4 DNA Ligase. NEB checking on whether the single one is available for purchase. Samples of this kit should be available from NEB.
HighPrep™ PCR Clean-up SystemMagBio Genomics Inc.Catalog #AC-60005 : $117.88 per 50 mL. $0.047 per rxn.
Note: Most magnetic beads from most vendors can be used with the same protocol.
Total per Flongle run (1/2 rxns): $66.95
Total per MinION run: $133.84
Total per sample (Flongle: 480 samples): $0.139
Total per sample (Flongle: 960 samples): $0.07
Consumables
Eppendorf DNA LoBind 1.5mL tubes
10uL pipette tips
100-200uL pipette tips
Equipment
PCR tube rack
Vortex mixer
Mini centrifuge
PCR cleanup magnet
10uL Pipette
100uL Pipette
Hula mixer (Ebay): $200.00 (optional)
Quantus or Qubit Fluorometer (optional)
Protocol materials
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
In Materials and 5 steps
NEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S
Materials, Step 1
HighPrep™ PCR Clean-up SystemMagBio Genomics Inc.Catalog #AC-60005
Materials
Adapter Ligation
Adapter Ligation
Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.
LA - Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Quick T4 Ligase - NEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S
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 - Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down and place on ice.
EB - Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
SFB - Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
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
Spin down with a mini centrifuge for 00:00:05 .
5s
Incubate the reaction for 00:10:00 at room temperature. Pull the AmPure XP (AXP) from the freezer.
10m
Resuspend AMPure XP (AXP) magnetic bead stock by vortexing.
Add 20 µL (Flongle) or 40 µL (MinION) of resuspended beads to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature (or just place in a tube rack without the mixer). Pull the EB and SFB from the freezer.
5m
Spin down the sample for 00:00:05 and pellet on a magnet for 00:02:00 .
Keep the tube on the magnet, and pipette off the supernatant.
2m 5s
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 00:00:05 , then return the tube to the magnetic rack for 00:02:00 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 - Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
2m 5s
Repeat the previous step.
Spin down for 00:00:05 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.
5s
Remove the tube from the magnetic rack and resuspend the pellet in 7 µL Elution Buffer (EB). Incubate for 00:10:00 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.
10m
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 00:01:00 .
1m
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.
Quantification
Quantification
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.