Jul 28, 2023
Version 1
Whole genome amplification and long read sequencing using ONT V.1
- 1Biodiversity Program, Taiwan International Graduate Program, Academia Sinica and National Taiwan Normal University, Taipei, Taiwan;
- 2Department of Microbiology, Soochow University, Taipei, 111, Taiwan;
- 3Biodiversity Research Center, Academia Sinica Taiwan
Protocol Citation: YiChien Lee, Huei-Mien Ke, Isheng Jason Tsai 2023. Whole genome amplification and long read sequencing using ONT . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko5q5v5r/v1
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: June 26, 2023
Last Modified: July 28, 2023
Protocol Integer ID: 84001
Funders Acknowledgement:
Academia Sinica grant
Grant ID: AS-CDA-107-L01
National Science and Technology Council
Grant ID: 111-2628-B-001-021
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Abstract
A genome reference is a prerequisite for a complete understanding of the biology and evolution of a species. However, the major challenge remains to obtain high-quality DNA and RNA from the majority of organisms. Therefore, there is a need for having a protocol to bypassed the challenging stage of obtaining axenic cultures and limited the amount of DNA material from a limit individual. This protocol build on whole genome sequencing single nematode. With multiple displacement amplification (MDA) allows the genome from a single nematode to be amplified and can sequence with both long- and short-read sequencing. This protocol can be completed within two week including genome amplification and sequencing.Also, combines MDA and Oxford Nanopore sequencing and provides a cost- and labor-effective solution to generate complete assemblies in organisms with as little as 50 picograms of starting material and assemble a draft genome assembly.
Materials
REAGENTS
22 gauge needle
0.2ml PCR tube
DNA LoBind Tubes, Eppendorf, #EP0030108078
1X PBS (Phosphate-buffered saline )
REPLI-g Single Cell KitQiagenCatalog #150345
AMPure XPBechman CoulterCatalog #A63882
T7 Endonuclease I - 1,250 unitsNew England BiolabsCatalog #M0302L
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK109
Flow Cell Priming Kit Oxford Nanopore TechnologiesCatalog #EXP-FLP002
NEBNext Quick Ligation Module - 100 rxnsNew England BiolabsCatalog #E6056L
NEBNext FFPE DNA Repair Mix - 96 rxnsNew England BiolabsCatalog #M6630L
NEBNext Ultra II End Repair/dA-Tailing Module - 96 rxnsNew England BiolabsCatalog #E7546L
EQUIPMENT
Magnetic separator
Vortex mixer
Microfuge
Thermal cycler
Rotator mixer
Magnetic separator
Qubit
Gridion
[Optional] extraction and denature of genomic DNA
[Optional] extraction and denature of genomic DNA
Prepare DLB Lysis buffer:
33 µL REPLI-g DLB buffer
3 µL 1M DTT
Add 4 µL PBS buffer in 0.2ml PCR tube.
Transfer worm to the 0.2ml PCR tube with 4 µL PBS buffer.
Note
[Optional] You may cut the worm with 22 gauge needle. This may release the cells from the cuticles
Add 3 µL DLB Lysis buffer. Incubate sample on thermocycler at 65 °C for 00:10:00
10m
Add 3 µL of REPLI-g Stop Solution. Store the sample on ice.
Whole genome amplification
Whole genome amplification
8h 38m 30s
8h 38m 30s
Prepare REPLI-g polymerase master mix
9 µL Nuclease-free water
29 µL REPLI-g Reaction Buffer
2 µL REPLI-g DNA Polymerase
Note
The difference of these kits is the final amount of expected amplified templates from the
polymerase master mix (SC 40 µg, midi 40 µg, and mini 10 µg with 10ng DNA). We initially started with SC, but were only able to obtain midi and mini kits during the COVID pandemic. We have inserted this explanation and option in the publicised protocol (doi here).
Add 40 µL REPLI-g polymerase master mix to each reaction.
Note
set heated lid temperature to 70 °C )
Incubate sample at 30 °C for 08:00:00 .
Inactivate reaction at 65 °C for 00:03:00 .
Store amplified DNA at 4 °C .
8h 3m
Dilute 1 µL amplified DNA to 100X in dH2O.
Take 2 µL diluted amplified DNA for quantification with Qubit dsDNA High sensitivity assay.
Warm AMPure XP beads to Room temperature .
Resuspend the AMPure XP beads by vortexing.
Transfer the sample to a 1.5 ml Microtubes.
Add 90 µL of resuspended AMPure XP beads to the amplification reaction and thoroughly mixed.
Incubate the sample on a Tube Revolver for 00:10:00 at Room temperature .
10m
Keep the tube on the magnet until eluate is clear and colorless, and pipette off the supernatant. Wash the beads with 200 µL of freshly prepared 70 % (v/v) ethanol for 00:00:30 and remove the ethanol using a pipette and discard.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol.
30s
Allow to dry for 00:05:00
Note
Do not dry the pellet to the point of cracking.
5m
Remove the tube from the magnetic rack and resuspend pellet in 30 µL Nuclease-free water. Incubate for 00:20:00 at Room temperature .
20m
Spin down and place the tube back on the magnet until the eluate is clear and colorless.
Remove and retain 30 µL of eluate in a clean 1.5 ml Microtube.
Dilute 1 µL purified amplified DNA to 100X in dH2O.
Take 2 µL diluted purified amplified DNA for quantification with Qubit dsDNA High sensitivity assay.
T7 endo I digestion
T7 endo I digestion
1h 15m 30s
1h 15m 30s
For each reaction, mix the reagents in the following order in a clean 0.2 ml PCR tube. Add nuclease-free water until final volume of 25 µL . Mix gently by flicking the tube, and spin down.
| A | B | |
| 1.5 μg (Xμl) | amplified DNA | |
| 3μl | NEBuffer 2 | |
| 1.5μl | T7 Endonuclease I | |
| 25-Xμl | Nuclease-free water |
Incubate at 20 °C for 00:30:00 and 65 °C for 00:30:00 in thermal cycler.
1h
Resuspend the AMPure XP beads by vortexing.
Add 60 µL of resuspended AMPure XP beads to the amplification reaction and thoroughly mixed. Incubate the sample on a Tube Revolver for 00:10:00 at Room temperature .
10m
Keep the tube on the magnet until eluate is clear and colorless, and pipette off the supernatant.
Wash the beads with 200 µL of freshly prepared 70 % (v/v) ethanol for 00:00:30 and remove the ethanol using a pipette and discard. Spin down and place the tube back on the magnet. Pipette off any residual ethanol.
30s
Repeat step 21 again.
Allow to dry for 00:05:00 , but do not dry the pellet to the point of cracking.
5m
Remove the tube from the magnetic rack and resuspend pellet in 49 µL Nuclease-free water. Incubate for 20 minutes at RT. Spin down and place the tube back on the magnet until the eluate is clear and colorless.
Remove and retain 49 μl of eluate in a clean 1.5 ml microtubes.
Take 2 μl purified DNA for quantification with Qubit dsDNA High sensitivity assay.
ONT library prep and ONT sequencing
ONT library prep and ONT sequencing
ONT library prep were followed ONT Ligation sequencing gDNA (SQK-LSK109) protocol.