TITLE: FindingNemo (v.kit14): A Toolkit for DNA Extraction, Library Preparation and Purification for Ultra Long Nanopore Sequencing
AUTHORS: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
This collection of protocols is designed to enable ultra-long (UL) reads on Nanopore sequencers. It is split into five sections dealing with ultra-high molecular weight (UHMW) DNA:
Extraction
QC 
Library preparation 
Nemo clean-up using glass beads and Hexamminecobalt(III) Chloride, aka. CoHex. 
Flowcell priming, library loading and run

We have tested and optimised the full protocol in human cell lines. 

Various options are available for each of the steps and we hope that the components here will be useful to the community and provide a long-read toolkit.

 


[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.

[GUIDELINES]
Acknowledgements

This protocol 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

Results

Below are some results from human genomic DNA runs on PromethION R10.4.1 flowcells after 24 hours of run, to give an idea of the range of output produced from 6 millions of input cells:
 



[STEPS]
SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
1. A DNA extraction protocol that yields clean and homogeneous UHMW DNA is essential for a good ultra-long (UL) sequencing output. We have routinely used the New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood (T3050) for UL library preparations, with the following modifications.

*This protocol has been optimised in human cell lines.*
SECTION: UHMW DNA QC
26. Two nucleic acid quantification methods, i.e., fluorometric (Qubit) and spectrophotometric (Nanodrop), can be used in parallel to assess both the quantity and quality of the extracted DNA. The quantification follows the published protocol by Koetsier and Cantor with slight modifications as follows.

An accurate measurement of DNA concentration is important as this will determine the optimum ratio of transposase (FRA) to DNA molecules at the library prep step. Also, the viscous nature of UHMW DNA requires that sample measurement represents all parts of the DNA solution.

A total of 9 µL  DNA is sampled from three different points of the solution: top, middle, and bottom part. Combine in a 2 ml tube.
 


SECTION: UHMW DNA QC
27. Add a glass bead and pulse vortex at full speed for a minute.
  

 


SECTION: UHMW DNA QC
28. Quantify with Qubit kit using the Jurkat genomic DNA as a standard.
 

SECTION: UHMW DNA QC
29. Quantify any RNA carry-over using the Qubit RNA Broad Range kit (optional).
 

SECTION: UHMW DNA QC
31. Whenever possible, the quality of extracted DNA sample should be analysed by method(s) that enable visual inspection of molecule length distribution such as:
Regular agarose gel electrophoresis
Pulsed-Field Gel Electrophoresis, e.g., using Pippin Pulse (Sage Science)
Agilent Bioanalyzer DNA
Agilent TapeStation DNA
SECTION: UL Library Prep
32. We are using the transposase-based library preparation kit from ONT, i.e. SQK-ULK114. 
SECTION: FindingNemo Library Clean-up
39. This section provides an alcohol-free purification of a nanopore DNA sequencing library from an UL protocol.
 
Add 3 borosilicate clean glass beads (3 mm diameter) into the sample in a 2 ml tube.
 

SECTION: FindingNemo Library Clean-up
40. Add 1 ml (1:1 volume) of 10 mM Hexamminecobalt(III) Chloride (CoHex) into the DNA solution. 
 

SECTION: FindingNemo Library Clean-up
41. Rotate the tube with a vertical rotator at 9 rpm for 3-5 minutes. 
  3 min 
 


SECTION: FindingNemo Library Clean-up
42. Invert the tube 3 times more by hand to ensure the DNA has precipitated and is tightly bound to the beads.
SECTION: FindingNemo Library Clean-up
43. Discard the supernatant. Take care not to disturb the DNA precipitated onto the beads.
SECTION: FindingNemo Library Clean-up
44. Wash the glass beads by gently adding 1 ml of PEGW buffer and gently invert 2-3 times. 
Incubate at Room temperature  for 3 min 
SECTION: FindingNemo Library Clean-up
45. Discard most of the wash buffer, again taking care not to disturb the DNA precipitate.
SECTION: FindingNemo Library Clean-up
46. Repeat wash with 500 µL .
SECTION: FindingNemo Library Clean-up
47. Discard the supernatant, taking care not to disturb the DNA precipitate. It isn't necessary to remove everything, a small volume of liquid can be left behind, i.e. dead volume. 
SECTION: FindingNemo Library Clean-up
48. Pulse-spin for 1 second in a mini centrifuge (or the shortest time possible) to collect residual wash buffer at the bottom. Remove last traces of buffer from under the glass beads with a fine pipette tip. 
SECTION: FindingNemo Library Clean-up
49. Quickly pour the DNA-bound beads into a new 2 ml tube that has been pre-aliquoted with 300 µL  or EB from the ULK114 kit.
 

SECTION: FindingNemo Library Clean-up
50. Incubate the library at 37 °C 30 min . Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P200 tip to aid elution.
SECTION: FindingNemo Library Clean-up
51. Insert a bead retainer into a clean 2 ml tube. Pour the beads from the previous step into the bead retainer and centrifuge at 10000 x g, 1 min 
SECTION: FindingNemo Library Clean-up
52. Incubate for at least 30 minutes at room temperature with regular pipette mixing.
Room temperature 30 min 
SECTION: FindingNemo Library Clean-up
53. Now - you have found Nemo!  
     

 

Store the library at 4 °Cor continue loading it to a flowcell.
SECTION: Flowcell Priming &amp; Library Loading
59. How to video from ONT:
YouTube Video: Loading a PromethION Flowcell
SECTION: Flowcell Priming &amp; Library Loading
55. Quantify 2-3 μl of the library sample using fluorometric method (Qubit DNA BR kit) or alternatively the spectophotometric method (Nanodrop).

 

SECTION: Flowcell Priming &amp; Library Loading
56. Prepare the library mix for loading as follow:

 

 

SECTION: Flowcell Priming &amp; Library Loading
57. Load the library on the flow cell and let tether for at least 30 minutes before starting the run.
30 min 
 

SECTION: Flowcell Priming &amp; Library Loading
58. Select the correct UL sequencing script based on the sequencing kit used (with the long winding down setting before every mux scan).
SECTION: Flowcell Nuclease Wash/Flush
60. Flush flowcell after every 20-24 hours or when active pores fall below 1000 (PromethION) using the flow cell wash kit (EXP-WSH004).

 

SECTION: Flowcell Nuclease Wash/Flush
61. Reprime the flow cell as before.

SECTION: Flowcell Nuclease Wash/Flush
62. Reload the library as before. 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
2. For each reaction, use 6 millions human cells, pellet and wash twice with cold PBS.
7000 x g, 1 min 

 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
3. Heat thermomixer to 56 °C . If available, change thermoblock into one that accommodates 5 ml Eppendorf LoBind tube; the following steps will be done in 5 ml tube. If not available, use 2 ml Eppendorf LoBind tube. 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
5. Resuspend cell pellet in 400 µL  and move cell suspension into a 5 ml Eppendorf LoBind tube. 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
4. Premix 400 µL  with 400 µL  and 40 µL 
from the kit reagents, vortex briefly to mix.

 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
6. Add 800 µL  to resuspended cells and mix by inverting 10 times.

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
7. Incubate on thermomixer at 56 °C  for 10 min 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
8. Add 20 µL , mix by careful pipetting 5 times using wide-bore P1000 tip, then invert-mix tube 5-10 times.

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
9. Incubate on thermomixer at 56 °C  for 10 min . 

 


SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
10. After lysis, add  300 µL  and mix by inverting 10 times. 

 


SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
11. Using a clean forceps add 3 Capture beads.

 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
12. Add 1.1 mL 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
13. Slowly manually invert until DNA has bound on the beads and no further compacting is observed, up to 25-30 inversions. Alternatively: invert in a vertical rotating mixer  

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
15. Remove supernatant
SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
16. Wash bound DNA with 750 µL  and carefully invert the tube 2-3 times. The DNA precipitate will contract more tightly around the beads. Remove Wash Buffer.

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
17. Repeat wash step once more.
SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
18. When most of the wash buffer is removed pour beads in the Monarch Bead retainer sitting in a Monarch Collection tube. Remove residual wash buffer by pulse spin as short as possible.

 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
14. Pre-aliquot 500 µL  into a clean 2 ml tube

 


SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
19. Immediately pour DNA-bound beads into the tube with pre-aliquoted Tris-HCl. 

 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
20. Incubate on thermomixer at 56 °C  for 10 min to elute DNA off the beads.

 


SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
21. In the mean time, place	the bead retainer back into the collection tube and spin in a minifuge for a few seconds to dry the bead retainer. Remove the collection tube with the wash buffer traces and place the dried bead retainer in a new DNA LoBind 2 ml tube.
SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
22. After elution, pour the beads with the eluted DNA into the bead retainer sitting in a 2 ml tube from the previous step.
SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
23. Centrifuge at 16000 x g, 1 min 
 


SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
24. Add 260 µL and using a wide bore pipette tip, carefully pipette eluate up and down 5-10 times for homogenization.

 

SECTION: UHMW DNA Extraction - Monarch Kit Direct Lysis
25. Incubate the samples at room temperature   

SECTION: UHMW DNA QC
30. Next, quantify DNA concentration and purity using Nanodrop (or other spectrophotometric devices), measuring three points in the solution: top, middle and bottom part. 

 

SECTION: UL Library Prep
33. Dilute or prepare DNA sample extracted from 6 million cells so concentration is between 20-40 ng/µl in a 2 ml tube. Mix to homogenise well and incubate at RT. Then, cool on ice. 

 

SECTION: UL Library Prep
34. Depending on the DNA volume in the previous step, prepare FRA dilution in a separate tube as in the table below:
 

SECTION: UL Library Prep
35. Mix the diluted FRA by quick vortexing. While keeping the tube on ice, add the diluted FRA to the DNA. Stir the reaction with the pipette tip whilst	expelling the diluted FRA to ensure an even distribution. Mix by gentle pipetting with a wide-bore pipette tip 25-30 times. 

 

SECTION: UL Library Prep
36. Incubate as follow:
Room temperature for 10 min 
75 °C  for 10 min  
on ice for 2 min 

SECTION: UL Library Prep
37. Add 5 µL to each sample with a regular pipette tip. Use a wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed.

SECTION: UL Library Prep
38. Incubate at   

SECTION: Flowcell Priming &amp; Library Loading
54. Prepare the priming mix as described in the ULK114 protocol using FCF and FTU.