TITLE: Whole genome amplification and long read sequencing using ONT 
AUTHORS: YiChien Lee, Huei-Mien Ke, Isheng Jason Tsai

[DESCRIPTION]
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]
SECTION: [Optional] extraction and denature of genomic DNA
1. Prepare DLB Lysis buffer: 
33 µL REPLI-g DLB buffer 
3 µL 1M DTT

SECTION: Whole genome amplification
6. Prepare REPLI-g polymerase master mix
9 µL Nuclease-free water
29 µL REPLI-g Reaction Buffer
2 µL REPLI-g DNA Polymerase

 


SECTION: [Optional] extraction and denature of genomic DNA
2. Add 4 µL PBS buffer in 0.2ml PCR tube.

SECTION: [Optional] extraction and denature of genomic DNA
3. Transfer worm to the 0.2ml PCR tube with 4 µL PBS buffer. 

 


SECTION: [Optional] extraction and denature of genomic DNA
4. Add 3 µL DLB Lysis buffer. Incubate sample on thermocycler at 65 °C for 10 min 

SECTION: [Optional] extraction and denature of genomic DNA
5. Add 3 µL of REPLI-g Stop Solution. Store the sample on ice.

SECTION: Whole genome amplification
7. Add 40 µL REPLI-g polymerase master mix to each reaction. 

 

Incubate sample at 30 °C for 480 min. 
Inactivate reaction at 65 °C for 3 min. 
Store amplified DNA at 4 °C.
 
SECTION: Whole genome amplification
8. Dilute 1 µL amplified DNA to 100X in dH2O. 
Take 2 µL diluted amplified DNA for quantification with Qubit dsDNA High sensitivity assay.

SECTION: Whole genome amplification
9. Warm AMPure XP beads to Room temperature. 
Resuspend the AMPure XP beads by vortexing.
SECTION: Whole genome amplification
10. Transfer the sample to a 1.5 ml Microtubes. 
Add 90 µL of resuspended AMPure XP beads to the amplification reaction and thoroughly mixed.

SECTION: Whole genome amplification
11. Incubate the sample on a Tube Revolver for 10 min at Room temperature.

SECTION: Whole genome amplification
12. 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 30 s and remove the ethanol using a pipette and discard. 
Spin down and place the tube back on the magnet. Pipette off any residual ethanol.

SECTION: Whole genome amplification
13. Allow to dry for 5 min 
 

SECTION: Whole genome amplification
14. Remove the tube from the magnetic rack and resuspend pellet in 30 µL Nuclease-free water. Incubate for 20 min at Room temperature.

SECTION: Whole genome amplification
15. 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.

SECTION: Whole genome amplification
16. 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.

SECTION: T7 endo I digestion
17. 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.

 



SECTION: T7 endo I digestion
18. Incubate at 20 °C for 30 minand 65 °C for 30 min in thermal cycler.

SECTION: T7 endo I digestion
19. 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 10 min at Room temperature.

SECTION: T7 endo I digestion
20. Keep the tube on the magnet until eluate is clear and colorless, and pipette off the supernatant.
SECTION: T7 endo I digestion
21. Wash the beads with 200 µL of freshly prepared 70 % (v/v) ethanol for 30 s and remove the ethanol using a pipette and discard. Spin down and place the tube back on the magnet. Pipette off any residual ethanol.

SECTION: T7 endo I digestion
22. Repeat step 21 again.
SECTION: T7 endo I digestion
23. Allow to dry for 5 min, but do not dry the pellet to the point of cracking.

SECTION: T7 endo I digestion
24. 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.

SECTION: T7 endo I digestion
25. Remove and retain 49 μl of eluate in a clean 1.5 ml microtubes.
SECTION: T7 endo I digestion
26. Take 2 μl purified DNA for quantification with Qubit dsDNA High sensitivity assay.
SECTION: ONT library prep and ONT sequencing
27. ONT library prep were followed ONT Ligation sequencing gDNA (SQK-LSK109) protocol.