Sep 02, 2020
Modified 1D Native Barcoding genomic DNA protocol from the Temperton Lab (University of Exeter)
- 1University of Chicago;
- 2University of Exeter;
- 3University of Southern California
- Thrash Lab
Protocol Citation: Michael Henson, Ben Temperton, Cameron Thrash 2020. Modified 1D Native Barcoding genomic DNA protocol from the Temperton Lab (University of Exeter). protocols.io https://dx.doi.org/10.17504/protocols.io.bixskfne
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 in our workspace and it is working.
Created: July 23, 2020
Last Modified: September 02, 2020
Protocol Integer ID: 39634
Keywords: MinION sequencing, 1D Native Barcoding genomic DNA protocol, NBE_9006_v103_revO_21Dec2016, Genome sequencing,
Abstract
Modified 1D Native Barcoding genomic DNA protocol (v. NBE_9006_v103_revO_21Dec2016 for SQK-LSK108) from the Temperton Lab (University of Exeter)
Materials
MATERIALS
Blunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S
NEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Ampure XP beads Beckman CoulterCatalog #A63881
Native Barcoding Kit 1D (EXP-NBD103)
Ligation Sequencing Kit 1DCatalog #SQK-LSK108
Qubit® dsDNA HS assay kit, 100 reactions Life TechnologiesCatalog #Q32851
g-TUBECovarisCatalog #520079
DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
Qubit™ dsDNA BR Assay KitThermo FisherCatalog #Q32853
DNA Fragmentation
DNA Fragmentation
Load 46 μL of genomic DNA into a Covaris g-TUBE.
Spin the g-TUBE for 1 minute in an Eppendorf 5424 centrifuge.
To achieve ~ XXX bp, spini the tube at 6000 x g.
Invert the tube and spin again for 1 minute.
Transfer volume to sterile 1.5 mL Eppendorf DNA LoBind tubes.
*Prior to the next step be sure to analyze your DNA for size, quantity, and quality.
Library Preparation
Library Preparation
Combine: 45 μL genomic DNA, 7 μL Ultra II End-prep reaction buffer, 3 μL Ultra II End-prep enzyme mix, 5 μL of Nuclease-free water.
Mix tube gently by flicking, then spin down.
Transfer volume to a new 0.2 mL PCR tube.
Incubate for 30 minutes at 20°C and 30 minutes at 65°C.
Add 60 μL of AmPure XP beads and mix by pipetting.
Incubate for 5 minutes at room temperature (RT), while flicking occasionally.
Spin down the sample and pellet on the magnet.
Pipette off supernatant while on the magnet, avoiding pellet.
Wash beads with 200 μL of 80% ethanol (EtOH). Do not disturb the pellet. Remove EtOH.
Repeat step 13
Spin down the tube and place the tube back in the magnetic rack.
Remove any residual ethanol.
Allow beads to briefly dry.
Remove the tube from the magnetic rack, and resuspend in 25 μL of prewarmed 55°C nuclease-free water.
Incubate for 2 minutes at 55°C.
Pellet beads on magnet until eluate is clear.
Pipette off 25 μL of eluate into 1.5 mL Eppendorf LoBind DNA tube.
Quantify 1 μL of eluate using Qubit HS dsDNA kit.
Thaw barcodes at RT.
Add 22.5 μL of end-prepped DNA, 2.5 μL Native barcode, and 25 μL of Blunt/TA ligase Master Mix.
Mix gently by flicking tube and spin down.
Incubate reaction mixture for 10 minutes at RT.
Add 50 μL of AMPure XP beads to the reaction mixture and mix by pipetting.
Incubate for 5 minutes at 55°C while occasionally flicking to mix.
Spin down the sample and pellet on the magnetic rack.
Wash beads with 200 μL of 80% EtOH. Do not disturb the pellet. Remove EtOH.
Pipette off supernatant, avoiding pellet.
Repeat step 31
Spin down the tube and place the tube back in the magnetic rack.
Remove any residual EtOH.
Allow beads to briefly dry.
Remove the tube from the magnetic rack, and resuspend in 25 μL of prewarmed 55°C nuclease-free water.
Incubate for 2 minutes at 55°C.
Pipette off 25 μL of eluate into 1.5 mL Eppendorf LoBind DNA tube.
Quantify 1 μL of eluate using the Qubit HS dsDNA kit.
Mix together 22.5 μL of end-prepped DNA, 2.5 μL of Native barcode, and 25 μL of Blunt/TA ligase Master Mix (in that order).
Mix by gently flicking.
Spin the tube down.
Incubate the reaction for 10 minutes at RT.
Add 50 μL of resuspended AMPure XP beads to the reaction and mix by pipetting.
Incubate at 55°C for 5 minutes while occasionally flicking to mix.
Spin down sample(s) and pellet on the magnetic rack.
Pipette off the supernatant.
Wash beads with 200 μL of 80% EtOH. Do not disturb the pellet. Remove EtOH.
Repeat step 47
Spin down the tube and place the tube back in the magnetic rack.
Remove any residual EtOH.
Allow the beads to briefly dry.
Remove the tube from the magnetic rack, and resuspend in 26 μL of nuclease-free water.
Incubate for 2 minutes at RT.
Pellet beads on the magnet until clear and colorless.
Transfer 26 μL into clear, sterile 1.5 mL Eppendorf DNA LoBind tube.
Quantify 1 μL of eluate using Qubit HS dsDNA kit.
Pool equimolar amounts of each barcoded sample into a 1.5 mL Eppendorf DNA LoBind tube (< 2 μg).
Quantify 1 μL of eluate using Qubit HS dsDNA kit.
Dilute pooled sample to 50 μL in Nuclease-free water.
Pooled Library Preparation
Pooled Library Preparation
Mix together 50 μL pooled barcoded samples sample, 20 μL barcode Adapter Mix (BAM), 20 μL Blunt/TA Master Mix, 10 μL Quick T4 DNA ligase.
Mix by flicking between each sequential addition.
Mix by flicking the tube.
Spin down the sample.
Incubate the reaction for 10 minutes at RT.
Add 40 μL of resuspended AMPure XP beads to the adapter ligation reaction and mix by pipetting.
Incubate for 5 minutes at RT, occasionally mixing by flicking.
Place on a magnetic rack and allow beads to pellet.
Pipette off supernatant.
Wash beads with 140 μL of ABB buffer.
Resuspend beads in ABB buffer by flicking the tube.
Pellet beads on the magnetic rack.
Remove residual ABB buffer.
Repeat step 68 - 71
Remove the tube from the magnetic tube and resuspend pellet in 15 μL of Elution buffer.
Incubate for 10 minutes at RT.
Pellet beads on the magnetic tube rack until the eluate is clear and colorless.
Pipet up and retain the 15 μL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube
Place the tube of eluate (library) on ice until required fro library loading
Use 1 μL of DNA library to Quantify using Qubit HS dsDNA assay kit.