TITLE: Strategic Savings in Ligation Sequencing: A Practical Nanopore Library Preparation Workflow
AUTHORS: Jie Hao Ou, Yin-Tse Huang

[DESCRIPTION]
This protocol introduces a cost-effective alternative for end repair and dA-tailing in DNA library preparation, particularly tailored for samples with an N50 of 3 kb. By employing a homebrew end repair reagent solution, this method replaces the use of the NEBNext® Ultra™ II End Repair/dA-Tailing Module recommended by Nanopore. Optimization of reagent quantities and a bead-free purification method are combined to achieve efficient adapter ligation while minimizing costs. Notably, an extended incubation time during adapter ligation enhances efficiency. This resource provides a strategic approach for researchers aiming to customize their sequencing workflows while achieving optimal results and substantial savings.

[GUIDELINES]
Optimized Homebrew Approach: This protocol features a unique homebrew end repair reagent solution as a cost-effective alternative to the Nanopore-recommended NEBNext® Ultra™ II End Repair/dA-Tailing Module. Adhere to the specified volumes meticulously to achieve successful results with your customized reagent.
Bead-Free Purification: The protocol employs a bead-free PEG/NaCl precipitation method for efficient purification. Ensure precise centrifugation to maintain DNA pellet integrity and maximize recovery rates.
Strategic Ligation Enhancement: An extended incubation period during adapter ligation enhances efficiency. Dedicate adequate time to this step to optimize adapter ligation results.

[STEPS]
SECTION: Homebrew End Repair/dA-Tailing
1.  Add the materials in the following table sequentially.
MaterialsQuantityDNA800 – 1600 ngH2OBring up to a volume of 43 ul2X PNK/Taq Buffer50 ulATP (25 mM)1 uldNTP (10 mM)5 ulTaq polymerase0.5 ul (2.5 U)T4 PNK0.5 ul (5 U)
 
 

 
 
SECTION: Homebrew End Repair/dA-Tailing
2. Incubate at 37 °C for30 min

 

SECTION: Homebrew End Repair/dA-Tailing
3. Incubate at 65 °C for 20 min

 

SECTION: Purification
4. For the subsequent purification, commercially available spin-column or magnetic bead-based methods can be used. 
Here, to save costs, we will use the PEG/NaCl precipitation method.
SECTION: Purification
5. Add 25 µL of 33.5% PEG 8000

SECTION: Purification
6. Add15 µL of NP


 


SECTION: Purification
7. Centrifuge at maximum speed (at least 8000 rpm) for30 min.

SECTION: Purification
8. Carefully remove the supernatant using a pipette.

 

SECTION: Purification
9. Add200 µLof 80% ethanol.
 


SECTION: Purification
10. Centrifuge at maximum speed (at least 8000 rpm) for2 min

SECTION: Purification
11. Carefully remove the supernatant using a pipette.
SECTION: Purification
12. Add200 µL of 99.5% ethanol.

SECTION: Purification
13. Centrifuge at maximum speed (at least 8000 rpm) for 2 minutes.
SECTION: Purification
14. Carefully remove the supernatant using a pipette.
SECTION: Purification
15. Wait for approximately10 min to ensure complete ethanol evaporation.

SECTION: Purification
16. Add25 µL of elution buffer.

 

SECTION: Adapter ligation
17. When using LNB provided by Nanopore
ReagentVolumeDNA400 ng (for samples with N50 of 3 kb)H2OBring up to a volume of 15 μlLigation Buffer (LNB)6.25 μlT4 Ligase2.5 μlLigation Adapter1.25 μl

When using homemade 4X Quick Ligase Buffer
ReagentVolume1DNA400 ng (for samples with N50 of 3 kb)2H2OBring up to a volume of 15.25 μl3Ligation Buffer (LNB)6.25 μl4T4 Ligase2.5 μl5Ligation Adapter1 μl
 

 

 

SECTION: Adapter ligation
18. Incubate at room temperature for60 min.
 



SECTION: Adapter ligation
19. Add3 µL of NP

 

SECTION: Adapter ligation
20. Centrifuge at maximum speed (at least 8000 rpm) for at least 30 min (If a higher recovery rate is needed, centrifuging for up to one hour is also possible).
 


SECTION: Adapter ligation
21. Carefully remove the supernatant using a pipette.

 

SECTION: Adapter ligation
22. Add 200 uL of LFB wash buffer
 


SECTION: Adapter ligation
24. Carefully remove the supernatant using a pipette.
SECTION: Adapter ligation
25. Repeat the above washing step twice.
SECTION: Adapter ligation
26. Add 20 µL of EB buffer (provided by the Ligation Sequencing Kit).
 


SECTION: Adapter ligation
23. Centrifuge at maximum speed (at least 8000 rpm) for2 min