TITLE: Soil Metagenome ONT
AUTHORS: Robert S James, Gaetan Benoit, Sebastian Raguideau, Georgina Alabone, Christopher Quince

[DESCRIPTION]
This protocol describes the sample collection to sequence acquisition workflow for Oxford Nanopore long-read sequencing of a complex soil sample using a ligation sequencing kit kit LSK-114 and R10.4.1 FLO-PRO114M flowcells. 

[BEFORE_START]
Dilute the concentrated SEWS-M solution with 100 mL of 100 % (v/v) EtOH before use.
Prepare 10 mL of fresh 80 % (v/v)  EtOH.



[GUIDELINES]
Fully equilibriate Ampure XP SPRI beads to Room temperature before use.
Fully equilibriate Qubit solution to Room temperature before use.
Fully equilibriate Tape station screen tape and reagents to Room temperature before use.
Over drying DNA bound to Ampure XP SPRI beads can reduce   recovery.
Additional time must be given for the Ampure XP beads to clear from the adapter ligation reaction due to the high viscosity of the solution. Failure to provide adequate time for the supernatant to clear can result in     loss.







[STEPS]
SECTION: 1. Soil sample collection and storage
1. Collect approximately 15 g of soil using a sterile soil corer or similar device and transfer to a sterile soil sieve and collection plate.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
107. Complete the requested sequencing parameters on the MinKNOW GUI and commence sequence acquisition.
SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
106. Incubate the flow cell for 30 min at Room temperature.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
105. Close the sample port valve by rotating it anti-clockwise and ensure the light shield is located securely.
SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
104. Aspirate 200 µL of   using the P 1000 and pipette then load into the PromerthION flow cell through the exposed sample port. Ensure no air bubbles are present in the end of the pipette and loading channel prior to loading the final library. Load the entire library by manually reducing the set pipette volume.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
103. Aspirate 168 µL of the SB and LIB mixture and dispense directly into the   with enough force to mix all reagents. Proceed directly to the next step.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
102. Set a P1000 pipette and tip to 200 µL and mix the Sequencing Buffer (SB) and library loading beads (LIB) mixture by pipetting up and down.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
101. Complete the flow cell flush by adding an additional 500 µL of the previously prepared Flow Cell Flush buffer to the PromethION flow cell through the sample port. 

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
100. Mix the Sequencing buffer (SB) by flicking then transfer 100 µL of SB to the 68 µL of LIB and return to ice. 

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
99. Resuspend the library loading beads (LIB) by pipetting then transfer 68 µL of LIB to a new 1.5 mL lo-bind tube and place on ice.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
98. Proceed with the protocol while incubating the flush buffer on the flow cell for 5 min at Room temperature.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
97. Load 500 µL of the prepared Flow Cell Flush Buffer through the exposed sample port of the PromethION flow cell. Care must be taken not to introduce air to the loading channel and flow cell during this step.

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
96. Remove ~20 µL of storage buffer from the sample loading port using a P1000 pipette and tip. Draw back the buffer by placing the end of the pipette tip into the sample port and manually increasing the set pipette fill volume. 

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
95. Rotate the sample port valve clockwise to expose the sample port.
SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
94. Locate the PromethION flow cell securely in the sequencing device  by.
SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
93. Add the following reagents to a 1.5 mL lo-bind Eppendorf tube, mix by pipetting up and down then store on ice.

1170 µL 
30 µL 

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
92. Briefly mix all solutions by flicking then spin down the tubes to collect the contents and return to ice.
SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
91. Thaw the Sequencing Buffer (SB), library beads (LIB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) at Room temperature. 

SECTION: 5. PromethION Flow cell priming, loading and sequence acquisition
90. Remove PromethION flow cell(s) from the fridge and allow to equilibriate to Room temperature for approximately 20 min.

SECTION: 4. Adapter ligation
89. Aspirate 32 µL of   and transfer to a new 1.5 mL lo-bind Eppendorf tube. Store the final library on ice until ready to load. Proceed directly to the next step in this protocol.

SECTION: 4. Adapter ligation
88. Quantify the [DNA] and fragment size distribution of a 10 % (v/v) dilution of the   in molecular grade water using a Qubit 4 fluorometer, 1x HS assay kit and Tape station Genomic screen tape.  

SECTION: 4. Adapter ligation
87. Replace the   on the magnetic rack and allow the solution to clear.

SECTION: 4. Adapter ligation
86. Transfer the   to a heat block at 37 °C and incubate for 10 min. 

SECTION: 4. Adapter ligation
85. Remove the tube from the magnetic rack and add 33 µL of ONT Elution buffer (EB) across the SPRI beads. Resuspend the beads by flicking then briefly spin to collect a homogenous solution at the bottom of the tube.

SECTION: 4. Adapter ligation
84. Allow the Ampure XP SPRI beads to air dry for 30 s.

SECTION: 4. Adapter ligation
83. Aspirate and discard residual LFB using a P 10 pipette and tip.
SECTION: 4. Adapter ligation
82.   Aspirate and discard the supernatant, then briefly centrifuge the   to collect residual LFB at the bottom of the tube then return the   to the magnetic rack.

SECTION: 4. Adapter ligation
81. Repeat steps 78 - 80 then continue to step 82.
SECTION: 4. Adapter ligation
80. Briefly centrifuge the   to collect the solution at the bottom of the tube and return the   to the magnetic rack and allow the solution to fully clear.

SECTION: 4. Adapter ligation
79. Remove the   from the magnetic rack and add 250 µL of ONT long fragment buffer (LFB) across the beads then suspend the beads in the LFB by flicking the tube.

SECTION: 4. Adapter ligation
78. Aspirate and discard the supernatant without disturbing the beads.
SECTION: 4. Adapter ligation
77. Briefly centrifuge the   to collect the contents at the bottom of the tube, then place the   on a magnetic rack and allow the solution to clear. The   is viscous and will require additional time to fully clear (~5 min).

SECTION: 4. Adapter ligation
76. Place the    on an active rotational mixer and incubate at  .
SECTION: 4. Adapter ligation
75. Add 40 µL of resuspended Room temperature Ampure XP  SPRI beads to the   and mix by flicking until a homogenous solution has formed.

SECTION: 4. Adapter ligation
74. Incubate the   for 20 min at Room temperature.

SECTION: 4. Adapter ligation
73. Thoroughly mix the ligation reaction by flicking until a homogenous solution is achieved. Incomplete mixing can result in a reduced ligation efficiency.
SECTION: 4. Adapter ligation
72. Mix the following reagents in order in a 1.5 mL lo-bind Eppendorf tube. Mix the reaction by flicking between the addition of each reagent and place on ice.

60 µL
25 µL 
10 µL 
5 µL 

SECTION: 4. Adapter ligation
71. Thaw ONT Elution buffer (EB) and ONT long fragment buffer (LFB) at room temperature, mix by flicking, spin to collect and place on ice.

SECTION: 4. Adapter ligation
70. Thaw the ONT ligation buffer (LNB) at room temperature, mix by pipetting, then place on ice.

SECTION: 4. Adapter ligation
69. Mix the ONT ligation adapter (LA) and NEB Quick T4 ligase by flicking then briefly spin down to collect the contents in the bottom of the tube and place them on ice.

SECTION: 3. End prep and FFPE repair
68. Transfer 60 µL of   to a new 1.5 mL lo-bind Eppendorf tube and place on ice.

SECTION: 3. End prep and FFPE repair
67. Remove the   from the heat block and place directly into a magnetic rack and allow the solution to clear. 

SECTION: 3. End prep and FFPE repair
66. Remove the   from the magnetic rack and add 60 µL of molecular grade water to the beads. Suspend the beads by flicking the tube and place in a heat block at 37 °C for 10 min.
SECTION: 3. End prep and FFPE repair
65. Allow the   to air dry until the beads are satin in appearance (30 s). Avoid over drying the beads to the point of cracking. Restrict residual EtOH carryover.  

SECTION: 3. End prep and FFPE repair
64. Aspirate and discard the residual EtOH using a P10 pipette and tip.
SECTION: 3. End prep and FFPE repair
63. Briefly centrifuge the    to collect residual EtOH in the bottom of the tube and replace on the magnetic rack.

SECTION: 3. End prep and FFPE repair
62. Repeat steps 60 and 61 then continue to step 63.
SECTION: 3. End prep and FFPE repair
61. Aspirate and discard the supernatant without disturbing the beads.
SECTION: 3. End prep and FFPE repair
60. Add 200 µL of freshly prepared 80 % (v/v) EtOH across the beads and incubate at Room temperature  for > 30 s. 
SECTION: 3. End prep and FFPE repair
59. Aspirate and discard the supernatant without disturbing the beads.
SECTION: 3. End prep and FFPE repair
58. Briefly centrifuge the tube to collect the   (<1000 x g), then place the    on a magnetic rack and allow the solution to clear completely.

SECTION: 3. End prep and FFPE repair
57. Add 60 µL of resuspended Room temperature Ampure XP SPRI beads to the    and mix by flicking until a homogeneous solution has formed. Incubate the    on an active rotational mixer at  .
SECTION: 3. End prep and FFPE repair
56. Transfer 60 µL of   to a new and clean 1.5 mL lo-bind Eppendorf tube.

SECTION: 3. End prep and FFPE repair
55. Briefly centrifuge the    to collect the contents in the bottom of the tube and place into a thermal cycler with the heated lid set to 105 °C. Incubate the reaction using the following conditions:

20 °C 30 min 
65 °C 30 min 
4 °C 
SECTION: 3. End prep and FFPE repair
54. Add the following reagents to the   in the order listed. Mix the reaction by flicking between the addition of each reagent and return to ice.

3.5 µL 
3.5 µL 
2 µL 
3 µL 
SECTION: 3. End prep and FFPE repair
53. Transfer 2-2.5 µg of    to a new and clean 0.2 mL thin walled PCR tube and adjust the volume to 48 µL using molecular grade water, then place the    on ice.

SECTION: 2. DNA extraction and sample cleanup
52. Once the solution has cleared, Quantify 1 µL of   using a Qubit 4 fluorometer or similar device.

SECTION: 2. DNA extraction and sample cleanup
51. Incubate the   at 37 °Cfor 10 min then return the   to the magnetic rack.

SECTION: 2. DNA extraction and sample cleanup
50. Resuspend the beads in the water by flicking the tube until a homogenous solution has formed.
SECTION: 2. DNA extraction and sample cleanup
49. Remove the   from the magnetic rack and add 50 µL of molecular grade water to the beads.

SECTION: 2. DNA extraction and sample cleanup
48. Allow the beads to air dry for 30 s or until the beads are satin in appearance. Do not over dry the beads, avoid excess EtOH carry over.

SECTION: 2. DNA extraction and sample cleanup
47. Promptly remove the excess EtOH with a P10 pipette and tip without disturbing the beads.
SECTION: 2. DNA extraction and sample cleanup
46. Remove the   from the magnetic rack and briefly centrifuge at < 1000 x g to collect any residual EtOH then return the   to the magnetic rack.

SECTION: 2. DNA extraction and sample cleanup
45. Repeat steps 43 and 44 then continue to step 46.
SECTION: 2. DNA extraction and sample cleanup
44. Aspirate and discard the supernatant without disturbing the beads.
SECTION: 2. DNA extraction and sample cleanup
43. Gently add 200 µL of 80 % (v/v) EtOH across the beads and incubate on the magnetic rack for at least 30 s.

SECTION: 2. DNA extraction and sample cleanup
42. Once the   has cleared, aspirate and discard the supernatant without disturbing the beads.

SECTION: 2. DNA extraction and sample cleanup
41. Briefly spin down the   and place on a magnetic rack.

SECTION: 2. DNA extraction and sample cleanup
40. Incubate the   on a rotational mixer for 10 min at Room temperature.

SECTION: 2. DNA extraction and sample cleanup
39. Add 60 µL of Room temperature Ampure XP beads to the   and mix by flicking the tube until a homogeneous solution has formed.

SECTION: 2. DNA extraction and sample cleanup
38. Allow the   to equilibrate to Room temperature and add 1 µL RNase A at 20 mg/mL. Mix tube by flicking and then incubate at Room temperature  for 2 min.
SECTION: 2. DNA extraction and sample cleanup
37. Centrifuge the    at 14000 x g, 2 min . Discard the spin filter and retain the   eluate.

SECTION: 2. DNA extraction and sample cleanup
36. Incubate the   at 56 °C for 10 min with intermittent agitation.

SECTION: 2. DNA extraction and sample cleanup
35. Add 100 µL of 56 °C DES elution buffer to the binding matrix and agitate the spin filter until a slurry has formed.

SECTION: 2. DNA extraction and sample cleanup
34. Allow the binding matrix to air dry for 2 min.

SECTION: 2. DNA extraction and sample cleanup
33. Remove the spin filter from the catch tube and place it into a new and clean 1.5 mL lo-bind Eppendorf tube.

SECTION: 2. DNA extraction and sample cleanup
32. Centrifuge the   at 14000 x g, 2 min to collect excess EtOH.

SECTION: 2. DNA extraction and sample cleanup
31. Repeat steps 29 and 30 then continue to step 32.
SECTION: 2. DNA extraction and sample cleanup
30. Centrifuge the   at 14000 x g, 2 min then discard the flow through.

SECTION: 2. DNA extraction and sample cleanup
29. Add 500 µL of prepared SEWS-M buffer to the   bound binding matrix, close the lid securely and suspend the beads in the SEWS-M solution by flicking the tube.


 

SECTION: 2. DNA extraction and sample cleanup
28. Repeat steps 26 and 27 until all the binding matrix has been processed through the spin filter. 
SECTION: 2. DNA extraction and sample cleanup
27. Centrifuge the spin filter at 14000 x g, 2 min and discard flow through.

SECTION: 2. DNA extraction and sample cleanup
26. Remove the   from the rotational mixer and transfer 750 µL of sample to an MPbio spin filter using a wide bore P 1000 pipette tip. Return the   to the rotational mixer. 

SECTION: 2. DNA extraction and sample cleanup
25. Add 1000 µLof DES to a new and clean 1.5 mL lo-bind tube and place the tube into an active heat block set to 56 °C.
SECTION: 2. DNA extraction and sample cleanup
24. Incubate the   on a rotational mixer at  .

SECTION: 2. DNA extraction and sample cleanup
23. Gently decant the   directly into the binding matrix without disturbing the protein pellet, secure the lid and mix the   by inversion until a homogeneous solution has formed.

SECTION: 2. DNA extraction and sample cleanup
22. Homogenise the binding matrix immediately before use. Add 1000 µL of resuspended binding matrix to a clean 5 mL lo-bind tube.

 

SECTION: 2. DNA extraction and sample cleanup
21. Remove the   from the ice and centrifuge at 14000 x g, 2 min to pellet the protein precipitate.

SECTION: 2. DNA extraction and sample cleanup
20. Incubate the   on ice for 10 min to facilitate protein precipitation.

SECTION: 2. DNA extraction and sample cleanup
19. Decant the supernatant from the lysing matrix E tube into the pre chilled PPS without disturbing the pellet, then mix the   by inversion 10 times and place on ice.

SECTION: 2. DNA extraction and sample cleanup
18. Remove the lysing matrix-E tube from the ice and centrifuge at 14000 x g, 4 min to pellet the debris. 

SECTION: 2. DNA extraction and sample cleanup
17. Add 250 µL of protein precipitation solution (PPS) to a new and clean 1.5 mL lo-bind Eppendorf tube and chill on ice.

SECTION: 2. DNA extraction and sample cleanup
16. Repeat steps 14 and 15 then continue to step 17.
SECTION: 2. DNA extraction and sample cleanup
15. Incubate the   on ice for 5 min.

SECTION: 2. DNA extraction and sample cleanup
14. Place the matrix tube in a MPBio FastPrep instrument (or similar) and homogenise for 10 s at 5.0 ms  then return the   to ice.

SECTION: 2. DNA extraction and sample cleanup
13. Add 122 µL of MT buffer to the  , mix by inversion and incubate on ice for 5 min.

SECTION: 2. DNA extraction and sample cleanup
12. Resuspend the pellet in 978 µL of sodium phosphate buffer, then transfer the    to a new and clean lyzing matrix E tube.

SECTION: 2. DNA extraction and sample cleanup
11. Aspirate and discard the supernatant without disturbing the pellet.
SECTION: 2. DNA extraction and sample cleanup
10. Centrifuge at 5000 x g, 4 min to pellet the   .

SECTION: 2. DNA extraction and sample cleanup
9. Transfer 1000 µL of the   to a new and clean 1.5 ml lo-bind Eppendorf tube using a P1000 pipette and wide bore pipette tip.

SECTION: 2. DNA extraction and sample cleanup
8. Defrost the   for 15 min on ice   prior to commencing DNA extraction. 

SECTION: 1. Soil sample collection and storage
7. Snap freeze and store    at -80 °C for future use.

SECTION: 1. Soil sample collection and storage
6. Repeat step 5 until the total volume of sample has exhausted or the desired number of aliquots have been achieved. 
SECTION: 1. Soil sample collection and storage
5. Gently mix the bulk sample by hand to form a homogenous solution then aspirate 1000 µL of the total sample using a P1000 pipette and wide bore tip. Transfer the   into a new and sterile 2 mL cryovial and close the lid securely.

SECTION: 1. Soil sample collection and storage
4. Incubate for at least 240 min at room temperature or 4 °C  240 min with occasional mixing.

SECTION: 1. Soil sample collection and storage
3. Use a top pan balance, spatular and weigh boat to weigh 10-50 g of homogenised soil   and transfer to a 250 mL conical flask. Promptly suspend the soil in 100 mL of Zymo DNA/RNA shield for a final concentration of 100-500 mg/mL. 

SECTION: 1. Soil sample collection and storage
2. Homogenise the soil sample by passing it through the soil sieve and collecting the output on the collection plate below the sieve. The use of a sterile plate sealer can be used to facilitate sieve homogenisation.