Oct 28, 2020

Public workspaceLong Read Viromics Amplification Library Preparation (VirION 2)

DOI
dx.doi.org/10.17504/protocols.io.5yug7ww
  • 1The Ohio State University;
  • 2University of Exeter
  • Sullivan Lab
Marie Burris
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DOI: dx.doi.org/10.17504/protocols.io.5yug7ww
Protocol Citation: Marie Burris, Natalie Solonenko, Olivier Zablocki, Ben Temperton 2020. Long Read Viromics Amplification Library Preparation (VirION 2). protocols.io https://dx.doi.org/10.17504/protocols.io.5yug7ww
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: July 29, 2019
Last Modified: October 28, 2020
Protocol Integer ID: 26356
Guidelines
Use DNA from "preparation of extracted DNA for long read library prep" as starting material for this protocol.

Use DNA LoBind tubes throughout this entire protocol (except for PCR tubes).
ReagentDNA LoBind TubesEppendorfCatalog ##022431021
Protocol materials
ReagentDNA LoBind TubesEppendorfCatalog ##022431021
ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
ReagentNEB Blunt/TA Ligase Master Mix Catalog #M0367
ReagentPCR Barcoding Expansion 1-12Catalog #EXP-PBC001
ReagentLA TaKaRa Hot StartTakara Bio Inc.Catalog #RR042A
Reagent1D Genomic DNA by LigationCatalog #SQK-LSK109
Sample Concentration
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
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 buffer
  • 2 ul NEB FFPE DNA enzyme mix
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S

ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S

Note
We recommend making 0.2 reactions worth of extra master mix to account for pipetting error.

In a PCR tube, add 12 ul master mix to 48 ul DNA from step 1.
Using a thermal cycler, incubate at 25 C for 5 minutes and 65 C for 5 minutes.
Temperature25 °C Duration00:05:00
Temperature65 °C Duration00:05:00




Clean up using AMPure XP beads (1:1 sample:beads) and elute in 31ul nuclease-free water into a 1.5mL tube.
  • See "Ampure Bead Clean up For HMW DNA" protocol.
  • For final elution step, incubate at Temperature55 °C Duration00:02:00
Reagent Ampure XP beads Beckman CoulterCatalog #A63881



Use 1ul to assess DNA concentration with Qubit.
Adapter ligation
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)

ReagentNEB Blunt/TA Ligase Master Mix Catalog #M0367


ReagentPCR Barcoding Expansion 1-12Catalog #EXP-PBC001


Incubate at room termperature for 10 minutes.
TemperatureRoom temperature Duration00:10:00


Clean up using AMPure XP beads (1:0.4 sample:beads) and elute in 15ul Nuclease-free water into a PCR tube.
  • See "Ampure Bead Clean up For HMW DNA" protocol.
  • For final elution step, incubate at Temperature55 °C Duration00:02:00
Use 1ul to assess DNA concentration with Qubit.
PCR Amplification
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 dNTPs
  • 10 ul 10x reaction buffer
  • 1 ul LA TaKaRa enzyme
  • 66 ul water

ReagentLA TaKaRa Hot StartTakara Bio Inc.Catalog #RR042A

Note
We recommend making 0.2 reactions worth of extra master mix to account for pipetting error.

Add 2 ul of your chosen barcode from Oxford Nanopore PCR Barcoding Expansion 1-12 kit to 5 ul of DNA from previous step.

Note
Be sure to use different barcodes for any samples that will be sequenced together.

Add 93 ul of master mix to the barcode - DNA mix
Use the following thermocycler conditions to amplify the library using minimum of 15 cycles.
  • repeat steps 2-4 of this sub-step 15-25 times

1.)Temperature94 °C Duration00:01:00

2.)Temperature94 °C Duration00:00:30
3.)Temperature62 °C Duration00:00:30
4.)Temperature68 °C Duration00:16:00 for 20kb

5.)Temperature72 °C Duration00:16:00 for 20kb

Clean up using AMPure XP beads (1:0.5 sample:beads) and elute in 20ul Nuclease-free water.
  • See "Ampure Bead Clean up For HMW DNA" protocol.
  • For final elution step, incubate at Temperature55 °C Duration00:02:00
Check the concentration (we recommend Qubit) and purity (NanoDrop) of your DNA, and run on a Genomic DNA TapeStation to assess library size.
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.

Reagent1D Genomic DNA by LigationCatalog #SQK-LSK109