FindingNemo Library 1: Modified ULK001
Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose
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 protocols we tested are based on ONT's rapid kit, i.e., SQK-ULK001 , a transposase based adapter ligation kit.
Modified ULK001 protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA and is our recommended route for best output as it is also the most-cost effective.
Transposase-based reaction is done in a large volume of up to 1 ml.
The working principle of the ULK001 protocol is shown in the diagram below:
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
Steps
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-F).
We follow the original 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)
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.
| 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 the dilution buffer (FDB) 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) | FDB (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 0h 10m 0s
70°C 0h 5m 0s
Room temperature 0h 10m 0s
Adapter Ligation
Add the corresponding volume of sequencing adapter (RAP-F) as in the table below.
| A | B | C | D | E |
|---|---|---|---|---|
| Cell No. (million) | Approx. DNA amount (μg) | FRA (μl) | RAP-F (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 |
Incubate for 30 minutes at 23oC.
23°C 0h 30m 0s
