TITLE: Long Read Viromics Amplification Library Preparation (VirION 2)
AUTHORS: Marie Burris, Natalie Solonenko, Olivier Zablocki, Ben Temperton

[STEPS]
?. [Sample Concentration ]
Prepare 48 ul of sample to use as input for the next step. Sample should have between 1ng - 100 ng of DNA for reliable library success. See "Preparation of extracted DNA for long-read library prep" concerning preparation of DNA for this protocol.
?. [DNA repair, End repair and dA tailing]
Prepare reaction mix using the following, making enough master mix for the total number of samples you are working with.3.5 ul NEB Ultra II End Repair/dA Tailing reaction buffer 3 ul NEB Ultra II End Repair/dA Tailing enzyme mix 3.5 ul NEB FFPE DNA repair buffer2 ul NEB FFPE DNA enzyme mix
We recommend making 0.2 reactions worth of extra master mix to account for pipetting error.
?. [DNA repair, End repair and dA tailing]
Using a thermal cycler, incubate at 25 C for 5 minutes and 65 C for 5 minutes.
25 °C
65 °C
?. [DNA repair, End repair and dA tailing]
Clean up using AMPure XP beads (1:1 sample:beads) and elute in 31ul nuclease-free water into a 1.5mL tube.For final elution step, incubate at
55 °C
?. [DNA repair, End repair and dA tailing]
Use 1ul to assess DNA concentration with Qubit.
?. [Adapter ligation]
Add the following to 30 ul DNA from previous step:  50 ul NEB Blunt/TA ligase 20 ul BCA (from Oxford Nanopore PCR Barcoding Expansion 1-12 kit)
?. [Adapter ligation]
Incubate at room termperature for 10 minutes.
0 Room temperature
?. [Adapter ligation]
Clean up using AMPure XP beads (1:0.4 sample:beads) and elute in 15ul Nuclease-free water into a PCR tube.For final elution step, incubate at
55 °C
?. [Adapter ligation]
Use 1ul to assess DNA concentration with Qubit.
?. [PCR Amplification]
Prepare reaction mix using the following from the LA TaKaRa Hot Start kit, making enough master mix for the total number of sample you are working with.16 ul dNTPs10 ul 10x reaction buffer1 ul LA TaKaRa enzyme 66 ul water
We recommend making 0.2 reactions worth of extra master mix to account for pipetting error.
?. [PCR Amplification]
Use the following thermocycler conditions to amplify the library using minimum of 15 cycles.1.)2.)3.)4.)5.)
94 °C
94 °C
62 °C
68 °C
[for 20kb]
72 °C
[for 20kb]
?. [PCR Amplification]
Clean up using AMPure XP beads (1:0.5 sample:beads) and elute in 20ul Nuclease-free water.For final elution step, incubate at
55 °C
?. [PCR Amplification]
Check the concentration (we recommend Qubit) and purity (NanoDrop) of your DNA, and run on a Genomic DNA TapeStation to assess library size.
?. [PCR Amplification]
Add  2 ul of your chosen barcode from Oxford Nanopore PCR Barcoding Expansion 1-12 kit to 5 ul of DNA from previous step.
Be sure to use different barcodes for any samples that will be sequenced together.
?. [DNA repair, End repair and dA tailing]
In a PCR tube, add 12 ul master mix to 48 ul DNA from step 1.
?. [PCR Amplification]
Add 93 ul of master mix to the barcode - DNA mix
?. [PCR Amplification]
The amplified libraries should be used as input for the Oxford Nanopore 1D Genomic DNA by Ligation (SQK-LSK109) protocol, beginning with the "DNA repair and end-prep" step. Libraries with different barcodes can be pooled at your desired ratio before beginning. We recommend loading 10 - 100 fmol of DNA into the flow cell.