Nov 21, 2023

Public workspaceStrategic Savings in Ligation Sequencing: A Practical Nanopore Library Preparation Workflow V.1

This protocol is a draft, published without a DOI.
  • 1National Chung Hsing University, Taichung;
  • 2Kaohsiung Medical University
Jie Hao Ou
Open access
Protocol Citation: Jie Hao Ou, Yin-Tse Huang 2023. Strategic Savings in Ligation Sequencing: A Practical Nanopore Library Preparation Workflow. protocols.io https://protocols.io/view/strategic-savings-in-ligation-sequencing-a-practic-cypvxvn6
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: August 15, 2023
Last Modified: November 21, 2023
Protocol Integer ID: 86485
Keywords: DNA library preparation, Nanopore, Cost-effective ligation, Bead-free purification
Abstract
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
  1. 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.
  2. 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.
  3. Strategic Ligation Enhancement: An extended incubation period during adapter ligation enhances efficiency. Dedicate adequate time to this step to optimize adapter ligation results.
Materials
1. 4X Quick Ligase Buffer

ABC
AB
1H2O70 ml
2Tris-Base2.42 g (200 mM)
3MgCl2・6H2O0.8 g (40 mM)
4DTT0.6 g (40 mM)
5PEG 800020 g (20% w/w)
6HClAdjust pH to 7.6
7H2Obring the volume up to 100 mL
Add the materials in the following table sequentially
2. 33.5% (w/v) PEG 8000
3. NP (5M NaCl + 6.7% PEG8000)
ABC
1PEG 80003.35 g (6.7%)
2NaCl14.61 g (5M)
4. 80% (v/v) ethanol
5. 99.5% ethanol
6. Elution buffer (1X TE, pH=8.0)
7. LFB wash buffer
ABC
133.5% (w/v) PEG 800080 uL
2NP40 uL
3H2O320 uL


Safety warnings
Attention
1. Ethanol Flammability: Ethanol is highly flammable. Use caution when handling and storing ethanol solutions. Work in well-ventilated areas away from open flames, sparks, or heat sources.
Homebrew End Repair/dA-Tailing
Homebrew End Repair/dA-Tailing
50m
Add the materials in the following table sequentially.
MaterialsQuantity
DNA1600 ng (for samples with N50 of 3 kb)
H2OBring up to a volume of 68 ul
4X Quick Ligase Buffer25 ul
ATP (25 mM)1 ul
dNTP (10 mM)5 ul
Taq polymerase0.5 ul (2.5 U)
T4 PNK0.5 ul (5 U)
Note
1. Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing.

Incubate at Temperature37 °C forDuration00:30:00

Note
1. T4 PNK will add phosphate to the 5' ends of both strands of DNA

30m
Incubate at Temperature65 °C for Duration00:20:00

Note
T4 PNK will be inactivated, and Taq polymerase will begin repairing DNA ends and adding A-tails.

20m
Purification
Purification
30m
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.
Add Amount25 µL of 33.5% PEG 8000

AddAmount15 µL of NP


Note
1. Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing.


Centrifuge at maximum speed (at least 8000 rpm) forDuration00:30:00 .

30m
Carefully remove the supernatant using a pipette.

Note
Most of the time, the pellet is not visible to the naked eye, so pay attention to the orientation during centrifugation

AddAmount200 µL of 80% ethanol.
Note
Sometimes the pellet becomes more visible after adding ethanol.


Centrifuge at maximum speed (at least 8000 rpm) forDuration00:02:00

2m
Carefully remove the supernatant using a pipette.
AddAmount200 µL of 99.5% ethanol.

Centrifuge at maximum speed (at least 8000 rpm) for 2 minutes.
2m
Carefully remove the supernatant using a pipette.
Wait for approximatelyDuration00:10:00 to ensure complete ethanol evaporation.

10m
AddAmount25 µL of elution buffer.

Note
Optional: Measure the DNA concentration.
Normally, the DNA concentration should be >30 ng/μL.

Adapter ligation
Adapter ligation
30m
When using LNB provided by Nanopore
ReagentVolume
DNA400 ng (for samples with N50 of 3 kb)
H2OBring up to a volume of 15.25 μl
Ligation Buffer (LNB)6.25 μl
T4 Ligase2.5 μl
Ligation Adapter1 μl

When using homemade 4X Quick Ligase Buffer
AB
ReagentVolume
DNA400 ng (for samples with N50 of 3 kb)
H2OBring up to a volume of 14.25 μl
4X Quick Ligase Buffer6.25 μl
ATP (25 mM)1 ul
T4 Ligase2.5 μl
Ligation Adapter1 μl


Note
Note that the reagent amounts used in this step are about 1/4 of the manufacturer's recommended volume. This means that the ligation sequencing kit, which was originally designed for 6 uses, can now be utilized for 24 uses.
This adjustment is primarily due to our utilization of a bead-free purification method later on, which significantly enhances the recovery rate.

Incubate at room temperature forDuration01:00:00 .
Note
Despite the official recommendation of a 5-minute reaction, based on our own experience, a 60-minute reaction significantly improves adapter ligation efficiency.



1h
AddAmount3 µL of NP

Note
1. Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing.
2. Normally, PEG and NaCl buffer is required for DNA precipitation. However, it's evident that LNB buffer already contains a high concentration of PEG, so adding NP alone is sufficient for DNA precipitation. For more information, refer to: https://dx.doi.org/10.17504/protocols.io.7erhjd6.

Centrifuge at maximum speed (at least 8000 rpm) for at least Duration00:30:00 (If a higher recovery rate is needed, centrifuging for up to one hour is also possible).
Note
To prevent overheating, it is recommended to use a refrigerated centrifuge.
If a refrigerated centrifuge is unavailable, it is advised to perform centrifugation in two steps of 15 minutes each, with a 10-minute interval in between.


30m
Carefully remove the supernatant using a pipette.

Note
Most of the time, the pellet is not visible to the naked eye, so pay attention to the orientation during centrifugation

Add 200 uL of LFB wash buffer
Note
Note that in this step, use the LFB wash buffer instead of alcohol.
This is because the DNA now has motor proteins attached, and using alcohol could disrupt the motor proteins.


Centrifuge at maximum speed (at least 8000 rpm) forDuration00:02:00
2m
Carefully remove the supernatant using a pipette.
Repeat the above washing step twice.
Add Amount20 µL of EB buffer (provided by the Ligation Sequencing Kit).
Note
Optional: Measure the DNA concentration.
Normally, the DNA concentration should be >15 ng/μL.


Protocol references