Sep 08, 2021
FindingNemo Library 2: Modified RAD004
- 1University of Nottingham;
- 2University of British Columbia;
- 3University of Birmingham
Protocol Citation: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose 2021. FindingNemo Library 2: Modified RAD004. protocols.io https://dx.doi.org/10.17504/protocols.io.bxv4pn8w
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: September 02, 2021
Last Modified: September 08, 2021
Protocol Integer ID: 52892
Keywords: ultra-long sequencing, cohex, glass bead, nanopore, MinION, UHMW DNA, Monarch, Circulomics, phenol, SDS, CTAB, GM12878, Whatman, PromethION, Nanobind
Abstract
This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit.
This Modified RAD004 protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA, for when ULK001 is not accessible/available. Transposase-based reaction is done in a large volume of up to 1 ml.
The working principle of the RAD004 kit is shown in the diagram below:
Guidelines
Acknowledgements
The FindingNemo protocol series was developed by Inswasti Cahyani for the Long Read Club with significant contributions from John Tyson and Nadine Holmes, also Josh Quick, and continuous discussion and support of Matt Loose and Nick Loman. We would also like to thank Giron Koetsier (NEB) and Kelvin Liu (Circulomics) for lending their expertise and advance product trials.
Please follow on Twitter for latest updates and results:
@NininUoN
@mattloose
Materials
Chemicals/Compounds
- Tris-HCl pH 8.0Thermo ScientificCatalog #J22638-AE
- Nuclease-free WaterThermofisherCatalog #AM9920
- NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
- Magnesium ChlorideFisher ScientificCatalog #AC223210010
- Triton X-100Sigma AldrichCatalog #T8787-50ML
- GlycerolBio Basic Inc.Catalog #GB0232.SIZE.500ml
Made-up Buffer
4x MuA Buffer
- 100 mM Tris-HCl pH 8.0
- 40 mM MgCl2
- 440 mM NaCl
- 0.2% TritonX-100
- 40% Glycerol
Kits
- Rapid Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-RAD004
Disposables
- DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
- DNA LoBind 2.0ml PCR Clean Eppendorf TubesEppendorfCatalog #0030 108.078
- Wide-bore (or cut off) P1000 and P200 tips
Safety warnings
When handling phenol always wear PPE, keep a solution of 50% (w/v) PEG-400 nearby to treat the burn in the case of accidental splashes.
Before start
Things to observe at all times:
- Excessive and vigorous pipetting and vortexing should be avoided as these may shear the DNA.
- Make up buffers with nuclease-free water to avoid introducing nucleases to solutions.
- Avoid unnecessary heating and freezing; isolated DNA should be stable for storage in the fridge for months
Library Prep Notes
Library Prep Notes
Extracted UHMW DNA is often difficult to quantify due to its viscosity. However, accurate measurement of DNA concentration is crucial for calculating optimum ratio of the transposase enzyme to the DNA molecules.
We provide a protocol section for quantifying UHMW DNA in our 'FindingNemo' protocol master file.
Properly quantified DNA can then be processed for this library prep.
Both cell number and DNA concentration/amount are used to calculate the amount of transposase (FRA) and adapter (RAP).
We base this protocol on SQK-RAD004 but follow the SQK-ULK001 protocol for the optimum ratio of transposase amount to human genomic DNA:
6 μl FRA to 6 million human cells (or around 40 μg DNA)
For other species, the genome size has to be taken into account and the FRA to DNA ratio optimised, e.g., we had optimised a non-human cell line of 6.2 Gb genome at:
2.5 μl FRA to 1 million non-human cell (around 12-15 μg DNA)
The following table lists the replaced reagents between ULK001 and RAD004.
| A | B | C | |
| Reagent | ULK001 | RAD004 (modified) | |
| Dilution buffer | FDB | MuA buffer (see Materials) | |
| Transposase | FRA* | FRA^ | |
| Sequencing adapter | RAP-F | RAP |
(*) FRA enzyme in ULK001 kit
(^) FRA enzyme in RAD004 kit
Transposase Reaction
Transposase Reaction
In a 2 ml tube, dilute UHMW DNA to a concentration of around 50 ng/μl in a total volume of 750 μl (with water or elution buffer if required).
Mix well with a P1000 wide-bore tip.
Note
- DNA concentration can still range from 20-50 ng/μl to have optimum transposase reaction.
- If input DNA amount is less than 20 μg (1-3 million cells used), halve all the reaction volumes, i.e., 375 μl total DNA volume instead of 750 μl as in the table below.
- It is important to have as homogeneous DNA as possible at this step so the transposase can access and cut the DNA molecules with an even distribution. It is OK to pipette thoroughly but gently.
| A | B | C | D | E | |
| Cell No. (million) | Approx. DNA amount (μg) | Total DNA volume (μl) | DNA concentration (ng/μl) | Total reaction volume (μl) | |
| 6 | >20-40 | 750 | 20-50 | 1000 | |
| 5 | |||||
| 4 | |||||
| 3 | 5-20 | 375 | 500 | ||
| 2 | |||||
| 1 |
In a 1.5 ml tube, dilute the corresponding amount of transposase (FRA) with MuA buffer to a total volume of 250 μl (or 125 μl if doing half-reaction). More details in the table below.
| A | B | C | D | E | |
| Cell No. (million) | Approx. DNA amount (μg) | FRA (μl) | 4X MuA Buffer (μl) | Total reaction volume (μl) | |
| 6 | >20-40 | 6 | 244 | 1000 | |
| 5 | 5 | 245 | |||
| 4 | 4 | 246 | |||
| 3 | 5-20 | 3 | 122 | 500 | |
| 2 | 2 | 123 | |||
| 1 | 1 | 124 |
Mix the diluted FRA by vortexing for 2-3 seconds.
Using a P1000 wide-bore tip, add the diluted FRA to the DNA sample.
Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.
Mix thoroughly by gentle pipetting.
On ice
Incubate the reaction as follows:
23 °C 00:10:00
70 °C 00:05:00
Room temperature 00:10:00 at least
Note
It is important that the room temperature at the fragmentation step (first incubation step) does not fall below 20oC to ensure optimum reaction condition. The use of a water bath or heating block is recommended.
Adapter Ligation
Adapter Ligation
Add the corresponding volume of sequencing adapter (RAP) as in the table below.
| A | B | C | D | E | |
| Cell No. (million) | Approx. DNA amount (μg) | FRA (μl) | RAP (μl) | Total reaction volume (μl) | |
| 6 | >20-40 | 6 | 5 | 1000 | |
| 5 | 5 | 4.2 | |||
| 4 | 4 | 3.3 | |||
| 3 | 5-20 | 3 | 2.5 | 500 | |
| 2 | 2 | 1.7 | |||
| 1 | 1 | 0.8 |
Note
- Use a P1000 wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed.
- Tube inversion can be used to aid mixing.
Incubate for 30 minutes at 23oC.
23 °C 00:30:00