TITLE: Extraction and ONT MinION Library Preparation of uHMW gDNA
AUTHORS: Kaylee S. Herzog, jfauver

[DESCRIPTION]
This custom protocol optimizes extraction, purification, and Oxford Nanopore Technologies (ONT) MinION library preparation for ultra-high molecular weight genomic DNA (uHMW gDNA) from parasitic nematodes. It can be used effectively with both low-input samples (e.g., a single adult hookworm) and high-input samples (e.g., a chunk of tissue from an Ascaris sp. adult).

Protocols on which this workflow is based:
Zymo® Quick-DNATM Magbead Plus Kit protcol
Oxford Nanopore Technologies® SQK-LSK-109 gDNA Ligation Sequencing protocol
Zymo® DNA Clean & ConcentratorTM Magbead Kit protocol (best-testing phase only)

[BEFORE_START]
Add 1,040 µL Zymo Proteinase K Storage Buffer to each tube of Zymo Proteinase K (20 mg) prior to use. The final concentration of Proteinase K is ~20 mg/ml. Store resuspended Proteinase K at -20°C after mixing.
For best results, allow AMPure XP beads (stored at 4°C) to come to RT prior to use.

[STEPS]
SECTION: Part 1: Ultra-HWM gDNA extraction | Zymo Quick-DNA HWM MagBead Plus Kit | ~3 hr
1. Set dry bath to 55 °C 

2. For each sample, add the following to a clean 1.5 mL microcentrifuge tube to create a master mix:
        95 µL  
        95 µL  
        10 µL  

2.1. Vortex the master mix gently to mix, then spin down and keep on ice
3. Using a new pipette tip or sterilized forceps, add one whole worm (or a piece of tissue) directly from tissue preservative to the bottom of a clean 1.5 mL microcentrifuge tube
 

4. Use a new   to grind and crush the tissue in the tube. Keep the pestle in the tube

5. Add 200 µL master mix (prepared in Part 1 Step 2) to each tube containing tissue and pestle

6. Continue using the pestle to grind the tissue within the master mix until fully homogenized. Remove the pestle, being careful to keep any tissue in the tube by wiping the pestle on the tube edges as it is removed
7. Close the tube and mix by inverting and flicking gently, then spin down briefly to recollect tissue and liquids
8. Incubate sample in dry bath at 55 °C for 150 min or until tissue solubilizes. During incubation, flick tube every 20 min to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
 


SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
9. Set dry bath to 37 °C

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
10. Add 400 µL   to each sample

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
11. Flick tubes to mix, then spin down briefly to recollect liquids
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
12. Add 33 µL   to each sample
 


SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
13. To ensure DNA binds to beads, mix on a rotator mixer at a low speed for 40 min at Room temperature. Spin down briefly before proceeding with the next step

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
14. Set sample tubes on a magnetic stand until beads have separated from solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand.
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
15. Add 500 µL   to each sample
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
16. Flick to mix initially, then mix on a rotator mixer at a low speed for 20 min at Room temperature. Spin down briefly before proceeding with the next step

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
17. Set sample tubes on a magnetic stand until beads have separated from the solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
18. Add 500 µL   to each sample
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
19. Flick to mix, then spin down briefly
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
20. Set sample tubes on a magnetic stand until beads have separated from solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
21. Add 900 µL   to each sample

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
22. Flick to mix, then spin down briefly
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
23. Transfer the entire sample (all liquid and beads) to a new clean 1.5 mL microcentrifuge tube
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
24. Set samples (now in new tubes) on a magnetic stand until beads have separated from the solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
25. Add 900 µL   to each sample
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
26. Flick to mix, then spin down briefly
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
27. Transfer the entire sample (all liquid and beads) to a new clean 1.5 mL microcentrifuge tube
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
28. Set samples (now in new tubes) on a magnetic stand until beads have separated from the solution, then remove and discard the supernatant. Leave sample tubes on the magnetic stand
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
28.1. Use a P10 pipette to remove any residual liquid from the bottom of the tube
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
29. Air dry the beads for up to 20 min and proceed to next step once beads are dry, but not over-dry
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
30. Add 50 µL    to each sample and flick gently several times to mix. Spin down briefly

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
31. Incubate in dry bath at 37 °C for 120 min. During incubation, flick tube every 20 min to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
32. Incubate on bench top at Room temperature overnight.

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
33. After overnight incubation, set tubes on a magnetic stand until beads have separated from solution, then move the supernatant (now containing eluted gDNA) to a new clean 1.5 mL microgentrifuge tube
 

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
33.1. Re-suspend beads in 20 µLof   in case there is no (or not enough) gDNA in final elution

SECTION: Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
34. Use 1 µL of final elution to quantify extraction via Qubit analysis

35. Use 1 µL of final elution to assess fragment size distribution via TapeStation
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
36. Set dry bath to 65 °C

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
37. Defrost the needed NEB DNA and End Repair reagents on ice (see Part 3 Step 38)
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
38. For each sample, add the following to a clean 0.2 mL PCR tube to create a master mix, pipetting 10–20 times between each addition to mix:
            3.5 µL   
            2 µL   
            3.5 µL  
           3 µL   

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
38.1. Keep master mix on ice
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
39. Add 12 µL of master mix (prepared in Part 3 Step 38) from the PCR tube directly into each 1.5 mL microcentrifuge tube containing extracted & purified uHWM gDNA (from Part 2). Mix all components by gently flicking, and spin tubes down to recollect liquids

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
40. Incubate samples at Room temperature for 10 min

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
41. Incubate samples in dry bath at 65 °C for 10 min

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
42. Add 4 volumes of   to each sample and mix well by flicking and inverting
 


SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
43. Spin samples down briefly and add 20 µL  
 


SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
44. Mix samples on rotating mixer at a low speed at Room temperature for 20 min

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
45. Briefly spin down samples and pellet on a magnetic stand (1–2 min) until the supernatant is clear and colorless. With the tubes still on the magnet, pipette off and discard the supernatant
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
46. Add 500 µL   and then remove from magnetic stand, and mix well by flicking and inverting

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
47. Briefly spin samples down briefly and transfer to magnetic stand to allow beads to pellet until solution is clear (1–2 min). With the tubes still on the magnet, pipette off and discard the supernatant
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
50. Air dry the beads for 10 min

 

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
51. Add 50 µL  

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
52. Manually agitate samples for 10 min by gently flicking/inverting (and occasionally spinning dow to recollect liquids)
 

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
53. Briefly spin samples down and pellet the beads on a magnet until the eluate is clear and colorless (1–2 min)
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
54. Remove and retain the 50 µL of eluate (containing repaired & end-prepped DNA) to a new clean 1.5 mL microcentrifuge tube

SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
55. Use 1 µL of final elution to quantify via Qubit assay

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
56. Set dry bath to 37 °C

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
57. Remove   from storage at 4 °C and allow them to come to Room temperature

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
58. Spin down   and  and place on ice

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
59. Thaw    at Room temperature, spin down, and mix by pipetting. Place on ice immediately after thawing and mixing

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
60. Thaw   at Room temperature, vortex to mix, spin down, and place on ice
SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
61. Thaw one tube each of    and   at Room temperature, vortex to mix, spin down, and place on ice

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
62. For each sample, add the following, in order, to a new clean 1.5 mL microcentrifuge tube, pipetting 10–20 times between each addition to mix:
            25 µL  
            10 µL  
            5 µL  
SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
62.1. Keep master mix on ice after mixing
SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
63. For each sample, prepare 1:3 SFB:LFB titrated wash mix by adding the following to a new clean 1.5 mL microcentrifuge tube, and then vortex to mix:
           125 µL    
            375 µL   

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
63.1. Keep titrated wash mix on ice after vortexing
SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
64. Pipette 40 µL of master mix (prepared in Part 4 Step 62) directly into entire volume of repaired and end-prepped gDNA from Part 3. Mix all components by gently flicking and spin tube down to recollect liquids

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
65. Incubate the reaction 15 min at Room temperature
 


SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
66. Resuspend    by vortexing and add 0.4X volume resuspended beads to each sample, then flick to mix
 

 

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
67. Mix on a rotator mixer at a low speed for 60 min at Room temperature

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
68. Spin down the sample and pellet on a magnetic stand. Keeping the tube on the stand, pipette off and discard the supernatant
SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
69. Wash the beads by adding 250 µL 1:3 SFB:LFB titrated wash mix (prepared in Part 4 Step 63). Flick the beads to resuspend, spin down, then return to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
70. Wash the beads by adding 250 µL 1:3 SFB:LFB titrated wash mix (prepared in Part 4 Step 63). Flick the beads to resuspend, spin down, then return to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
71. Spin down the beads and place them back on the magnetic rack. Use a P10 pipette to pipette of any residual liquid and allow beads to air-dry for 30 s to 2 min

 

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
72. Remove the tube from the magnetic stand and resuspend the beads in 15 µL  

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
73. Briefly spin down and incubate in dry bath at 37 °C for 120 min. During incubation, flick tube every 20 min to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
 


SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
74. Incubate on the bench top at Room temperature overnight

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
75. 1.After overnight incubation, pellet the beads on a magnet until the eluate is clear and colorless (at least 1 min)

SECTION: Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing &amp; NEBNext Companion Kits | ~3 hr + overnight incubation
76. Remove and retain the 15 µL of eluate (containing the prepared library) to a new clean 1.5 mL microcentrifuge tube

77. Use 1 µL of final elution to quantify library via Qubit analysis
 


SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
48. Add 500 µL   and then remove from magnetic stand, and mix well by flicking and inverting
SECTION: Part 3: DNA repair and end-prep | Zymo Clean &amp; Concentrator, ONT Ligation Sequencing, &amp; NEBNext Companion Kits | ~1.5 hr
49. Briefly spin samples down briefly and transfer to magnetic stand to allow beads to pellet until solution is clear (1–2 min). With the tubes still on the magnet, pipette off and discard the supernatant