Whole genome amplification and long read sequencing using ONT
YiChien Lee, Huei-Mien Ke, Isheng Jason Tsai
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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.
Steps
[Optional] extraction and denature of genomic DNA
Prepare DLB Lysis buffer:
33µL REPLI-g DLB buffer
3µL 1M DTT
Add 4µL PBS buffer in 0.2ml PCR tube.
Transfer worm to the 0.2ml PCR tube with 4µL PBS buffer.
Add 3µL DLB Lysis buffer. Incubate sample on thermocycler at 65°C for 0h 10m 0s
Add 3µL of REPLI-g Stop Solution. Store the sample on ice.
Whole genome amplification
Prepare REPLI-g polymerase master mix
9µL Nuclease-free water
29µL REPLI-g Reaction Buffer
2µL REPLI-g DNA Polymerase
Add 40µL REPLI-g polymerase master mix to each reaction.
Incubate sample at 30°C for 8h 0m 0s.
Inactivate reaction at 65°C for 0h 3m 0s.
Store amplified DNA at 4°C.
Dilute 1µL amplified DNA to 100X in dH2O.
Take 2µL 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 90µL of resuspended AMPure XP beads to the amplification reaction and thoroughly mixed.
Incubate the sample on a Tube Revolver for 0h 10m 0s at Room temperature.
Keep the tube on the magnet until eluate is clear and colorless, and pipette off the supernatant. Wash the beads with 200µL of freshly prepared 70% (v/v) ethanol for 0h 0m 30s and remove the ethanol using a pipette and discard.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol.
Allow to dry for 0h 5m 0s
Remove the tube from the magnetic rack and resuspend pellet in 30µL Nuclease-free water. Incubate for 0h 20m 0s at Room temperature.
Spin down and place the tube back on the magnet until the eluate is clear and colorless.
Remove and retain 30µL of eluate in a clean 1.5 ml Microtube.
Dilute 1µL purified amplified DNA to 100X in dH2O.
Take 2µL diluted purified amplified DNA for quantification with Qubit dsDNA High sensitivity assay.
T7 endo I digestion
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 25µL. 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 0h 30m 0sand 65°C for 0h 30m 0s in thermal cycler.
Resuspend the AMPure XP beads by vortexing.
Add 60µL of resuspended AMPure XP beads to the amplification reaction and thoroughly mixed. Incubate the sample on a Tube Revolver for 0h 10m 0s at Room temperature.
Keep the tube on the magnet until eluate is clear and colorless, and pipette off the supernatant.
Wash the beads with 200µL of freshly prepared 70% (v/v) ethanol for 0h 0m 30s and remove the ethanol using a pipette and discard. Spin down and place the tube back on the magnet. Pipette off any residual ethanol.
Repeat step 21 again.
Allow to dry for 0h 5m 0s, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend pellet in 49µL 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 were followed ONT Ligation sequencing gDNA (SQK-LSK109) protocol.
