TITLE: T-4 TICK TESTING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details standard operating procedure for tick testing.

[BEFORE_START]
BEFORE START

Check the DNA and RNA concentrations in each sample of total nucleic acid (TNA) extraction. 
If the concentrations are detectable, choose the sequencing approach following the table below. 
If DNA or RNA concentration is not detectable, prepare the library for the detectable one. 
Use sections gDNA PREPARATION and TNA PREPARATION for gDNA and TNA preparation, respectively, then subject the prepared gDNA and TNA to Section SEQUENCING LIBRARY PREPARATION.
 
                      DNA concentration (ng/ul)               < 1 ng/ul      1-10 ng/ul      > 10 ng/ul        RNA concentration (ng/ul)      < 4 ng/ul      TNA      DNA      DNA        4-20 ng/ul      TNA      TNA      TNA        >20 ng/ul      TNA      TNA      TNA   
 


[GUIDELINES]
OBJECTIVE

To outline the procedures for properly using the Oxford Nanopore Sequencing platforms (GridION or MinION Mk1C) to sequence gDNA and TNA extracted from collected tick samples.

SUMMARY/SCOPE

This SOP provides guidance on procedures of Oxford Nanopore sequencing to generate sequencing reads for downstream data analysis and pathogen detection.

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
 

MAINTENANCE OF EQUIPMENT

CAUTION ON RNA HANDLING:

RNases are very stable and difficult to inactivate and only minute amounts are sufficient to destroy RNA.
Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure.
Clean the work surfaces with RNA Zap to remove nucleases, then wipe the surfaces with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

HANDLING ENZYMATIC REACTIONS

Reagents containing enzymes should be handled on ice before mixed and transferred to the assigned activation temperature.

REFERENCES

REDI-NET Overview Summary

Double-stranded cDNA synthesis (NEB first and second strand cDNA synthesis protocols):
NEBNext Ultra II RNA First Strand synthesis manual E7771
NEBNext Ultra II Non-directional RNA Second Strand synthesis manual E6111 
ezdnase_PI

Oxford Nanopore Manufacturer's protocols:
Ligation sequencing gDNA - Native Barcoding Kit 96 V14 (SQK-NBD114.96)-minion. 
ligation-sequencing-gdna-native-barcoding-v14-sqk-nbd114-96-NBE_9171_v114_revG_15Sep2022-minion
ligation-sequencing-gdna-native-barcoding-v14-sqk-nbd114-96-NBE_9171_v114_revG_15Sep2022-gridion

APPENDICES

 
APPENDIX 1. FLOW CELL
 
APPENDIX 2. cDNA END-PREP MASTER MIX PREPARATION 
 
     Component      Volume for 1 reaction      Volume for n+1 reactions        cDNA sample      20 μl      20 μl        Nuclease-free   water       30 μl      … μl        Ultra II   End-prep reaction buffer      7 μl      … μl        Ultra II   End-prep enzyme mix      3 μl      … μl        Final total   volume      60 μl      … μl   
 
APPENDIX 3. EXPECTED OUTCOMES

The DNA or RNA inputs vs the sequencing read yields.
 
 
 


[STEPS]
SECTION: gDNA PREPARATION
1. 
When the RNA concentration of the sample is lower than the detectable range of the Qubit High Sensitivity Assay (<0.01 ng/ul), the sample is subjected to gDNA sequencing. The cDNA synthesis can be skipped.

SECTION: gDNA PREPARATION
2. When the DNA concentration >10 ng/ul, calculate the required volume of 200 ng DNA, then transfer the volume to a new 200μl PCR tube or a well of a 96-well PCR plate. Adjust the volume with nuclease-free water to a final volume of 20 µL.

SECTION: gDNA PREPARATION
3. Prepare 100 ng gDNA from positive control extraction in 20 µL nuclease-free water in a new 200μl PCR tube or a well of a 96-well PCR plate.

SECTION: gDNA PREPARATION
4. Transfer 20 µL negative control extraction to a new tube or a well of a 96-well PCR plate.

SECTION: gDNA PREPARATION
5. All samples are subjected to section SEQUENCING LIBRARY PREPARATION.
SECTION: TNA PREPARATION
6. To prepare TNA for sequencing both cDNA and gDNA, cDNA needs to be prepared separately and then mixed with TNA from the original sample.
SECTION: TNA PREPARATION
7. Prepare cDNA following section cDNA SYNTHESIS (positive control and negative control included) until step 40.
SECTION: TNA PREPARATION
8. Transfer 10 µL double-stranded cDNA of section cDNA SYNTHESIS step 40 to a new 200 μl PCR tube or a well of a 96-well PCR plate. Add 10 µL of TNA from the original sample to make the final volume 20 µL.

SECTION: TNA PREPARATION
9. Subject the 20 µL double-stranded cDNA/TNA mixture to section SEQUENCING LIBRARY PREPARATION.
 


SECTION: cDNA SYNTHESIS: DNase treatment
10. If RNA concentration > 20 ng/ul, calculate the required volume of 160 ng RNA, then transfer the volume to a new 200μl PCR tube or a well of a 96-well PCR plate. Adjust the volume with nuclease-free water to a final volume of 8 µL. If the concentration of the RNA < 20 ng/ul, directly transfer 8 µL of RNA to a new 200μl PCR tube or a well of a 96-well PCR plate. Keep the tube or plate on ice.

SECTION: cDNA SYNTHESIS: DNase treatment
11. Prepare 40 ng RNA from positive control extraction and adjust the volume to final 8 µL with nuclease-free water in a new 200μl PCR tube or a well of a 96-well PCR plate.

SECTION: cDNA SYNTHESIS: DNase treatment
12. Transfer 8 µL negative control extraction to a new tube or a well of a 96-well PCR plate.

SECTION: cDNA SYNTHESIS: DNase treatment
13. Remove contaminated DNA (~ 15 mins): Thaw total nucleic acid, 10x ezDNase Buffer, and DTT on the ice at Room temperature. Vortex 10x ezDNase Buffer and DTT briefly, spin down by centrifugation for 5 s, and place on ice. ezDNase is not frozen and should be placed on ice before use. Set up thermal cycler programs: 37 °C, 2 min, and 55 °C, 5 min.

SECTION: cDNA SYNTHESIS: DNase treatment
13.1. Mix the following components in an RNase-free tube or plate. For processing multiple samples, make a master mix for 10× ezDNase buffer and ezDNase with 10% overage. Aliquot the master mix into the wells of a 96-well plate, then add TNAs.
 
     Component      Volume        10× ezDNase Buffer      1 μl        ezDNase      1 μl        RNA from step 6.3.1       8 μl        Total volume      10 μl   
 
SECTION: cDNA SYNTHESIS: DNase treatment
13.2. Gently mix the samples then centrifuge the tube (Include a reaction for extraction positive control and negative control of each batch nucleic acid extraction).
SECTION: cDNA SYNTHESIS: DNase treatment
13.3. Incubate the sample for 2 min at 37 °C.
SECTION: cDNA SYNTHESIS: DNase treatment
13.4. Add 1 µL 100mM DTT into the reaction tube.

SECTION: cDNA SYNTHESIS: DNase treatment
13.5. Incubate the sample at 55 °C for 5 min to inactivate the enzyme.

SECTION: cDNA SYNTHESIS: DNase treatment
13.6. Chill the tube on ice to bring the sample to Room temperature, then spin down and place the tube on ice.

SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
15. Add the following reagents into the ezDNase-treated RNA from step 13.6. For processing multiple samples, make a master mix for the 60 μM Random Primer Mix and nuclease-free water with 10% overage. 
     Component      Volume        ezDNase   treated RNA       10 μl        60 μM Random   Primer       1 μl        Nuclease free   water      3 μl        Total volume       14 μl   
 
SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
16. Mix gently, spin down and incubate at 65 °C for 5 min. Chill on ice, spin down again and place on ice.

SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
17. Add the following components in the indicated order, if multiple reactions will be processed at the same time, make a master mix with a 10% overage:
 
     Component      Volume        NEBNext First   Strand Synthesis Reaction Buffer      4 μl        NEBNext First   Strand Synthesis Enzyme Mix      2 μl        Final total   volume      20 μl   
 
SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
18. Mix gently and spin down.
SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
19. Incubate the tube for 10 min at 25 °C followed by 15 min at 42 °C.

SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
20. Terminate the reaction by heating at 70 °C for 15 min.

SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
21. Place the tube on ice or pre-chilled freezer block.

SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
22. Continue immediately with the second strand synthesis reaction as described below.
SECTION: cDNA SYNTHESIS: Second strand cDNA Synthesis (~ 1hr)
23. Pipette the following components directly into the first strand reaction tube (with 20 µL mixture) on ice in the indicated order, if multiple reactions will be processed at the same time, make a master mix with a 10% overage:
 
     Component      Volume        5x NEBNext Second   Strand Synthesis Reaction Buffer      5 μl        NEBNext Second   Strand Synthesis Enzyme Mix      2.5 μl        Nuclease-free   water       22.5 μl        Final total   volume      50 μl   
 
SECTION: cDNA SYNTHESIS: Second strand cDNA Synthesis (~ 1hr)
24. Mix gently and centrifuge briefly.
SECTION: cDNA SYNTHESIS: Second strand cDNA Synthesis (~ 1hr)
25. Incubate at 16 °C for 60 min (heated lid set at ≤ 40 °C).

SECTION: cDNA SYNTHESIS: Second strand cDNA Synthesis (~ 1hr)
26. Proceed with cDNA purification or store the reaction mixture at -20 °C before the subsequent cDNA purification (the double-stranded cDNA is ready to be shipped to Gold Labs if necessary).

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
27. 
 
Resuspend the AMPure XP beads by vortexing.
SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
28. Transfer the sample (50 µL) to a clean 1.5ml low DNA binding tube.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
29. Add 40 µL of resuspended AMPure XP beads to the reaction and mix by flicking the tube.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
30. Incubate on a Hula mixer (rotator mixer) for 5 min at Room temperature.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
31. Spin down the sample and pellet on the magnet. Keep the tube on the magnet, and using a pipette, discard the supernatant.
SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
32. Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
33. Repeat the previous step X1.
SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
34. Spin down and place the tube back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
35. Remove the tube from the magnetic rack and resuspend the pellet in 13 µL nuclease-free water.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
36. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
37. Spin down and pellet beads on magnet until the eluate is clear and colorless.
SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
38. Remove and retain 11 µL of eluate into a clean 1.5ml low DNA binding tube.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
39. Optional: Analyze 1 µL of the purified double-stranded cDNA for quantity using Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit.

SECTION: cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
40. Subject 10 µL purified double-stranded cDNA for section  SEQUENCING LIBRARY PREPARATION.    
 


SECTION: SEQUENCING LIBRARY PREPARATION
41. Before starting, prepare fresh 70% ethanol in nuclease-free water sufficient for your samples
 (1 mLper sample). Program the thermal cycler or use a heat block for 96 well plate: 20 °C for 5 min and 65 °C for 5 min. Thaw Ultra II End-prep reaction buffer, NEBNext FFPE DNA Repair Buffer, Barcode Plate(from SQK-NBD114.96 Kit),and Blunt/TA Ligase Master Mix on ice. After fully thaw, mix by vortex, spin down briefly, and place on ice. Check that there is no precipitate present (the Blunt/TA Master Mix can sometimes form a precipitate). Spin down Ultra II End-prep enzyme mix and place on ice.


SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
42. Mix the following reagents in a 0.2ml PCR tube. To process 24 samples, prepare a master mix by multiplying gradients except for cDNA by 24 with a 10% overage. Aliquot the master mix into a 96-well plate, then add cDNA or TNA (see Appendix 2 for master mix preparation): 
 
     Component      Volume        DNA/TNA sample      20 μl        DNA control       0.5 μl    
  
SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
43. Mix gently by pipetting and spin down.
SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
44. Using a thermal cycler, incubate at 20 °C for 5 min and 65 °C for 5 min.

SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
45. Resuspend the AMPure XP beads by vortexing.
SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
46. Add 50 µL of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting (use an 8-channel pipette for reagent transfer of multiple samples).

SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
47. Incubate on a Hula mixer (rotator mixer) for 5 min at Room temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
48. Spin down the sample and pellet on a magnet (DynaMag-2 for 1.5ml tube and DynaMag-96 for PCR plate). Keep the tube on the magnet, and using a pipette, discard the supernatant.
SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
49. Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
50. Repeat the previous step X1.
SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
51. Spin down and place the tube back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.

SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
52. Remove the tube from the magnetic rack and resuspend the pellet in 12 µL nuclease-free water. Incubate for 2 min at Room temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
53. Pellet the beads on a magnet until the eluate is clear and colorless.
SECTION: SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
54. Remove and retain 11 µL of eluate into a clean 1.5ml low DNA binding tube.

SECTION: SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
55. Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:
 
   
      Component  Volume    End-prepped DNA  10 μl     Native   Barcode (pick one form Native Barcoding Expansion 1-96)   2 μl    Blunt/TA   Ligase Master Mix  12 μl    Final   total volume  24 μl  
  
SECTION: SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
56. Mix gently by flicking the tube and spin down.
SECTION: SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
57. Incubate the reaction for 20 min at Room temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
58. Add 3 µL of EDTA to each well and mix thoroughly by pipetting and spin down briefly.
 


SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
59. Pool every 24 barcoded samples 12 µL from each sample, (total 288 µL/pool) in a new 1.5ml low DNA binding tube.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
60. Resuspend the AMPure XP beads by vortexing.
SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
61. Add 518 µL (1.8x volume of the pooled library) of resuspended AMPure XP beads to the pooled library and mix by pipetting.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
62. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
63. Spin down the sample and pellet on a magnet. Keep the tube on the magnet for 5 min, and using a pipette, discard the supernatant.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
64. Keep the tube on the magnet and wash the beads with 700 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
65. Repeat the previous step X1.
SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
66. Spin down and place the tube back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
67. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL nuclease-free water. Incubate for 10 min at 37 °C temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
68. Spin down and pellet the beads on a magnet until the eluate is clear and colorless.
SECTION: SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
69. Remove and retain 35 µL of eluate into a clean 1.5ml low DNA binding tube.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
71. 
Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition. 
     Pooled   barcoded sample      30 μl        Native   Adapter (NA)      5 μl        NEBNext   Quick Ligation Reaction Buffer (5×)      10 μl        Quick   T4 DNA Ligase      5 μl        Final   total volume      50 μl   
 
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
72. Mix gently by flicking the tube, and spin down.
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
73. Incubate the reaction for 20 min at Room temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
74. Resuspend the AMPure XP beads by vortexing.
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
75. Add 90 µL of resuspended AMPure XP beads to the reaction and mix by pipetting.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
76. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
77. Place on the magnetic rack, allow beads to pellet and using a pipette, discard the supernatant.
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
78. Add 125 µL of the Short Fragment Buffer (SFB) to the beads. Close the tube lid and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow beads to pellet and using a pipette, discard the supernatant.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
79. Repeat the previous step X1.
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
80. Spin down and place the tube back on the magnet. Using a pipette, remove any residual supernatant.
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
81. Remove the tube from the magnetic rack and resuspend the pellet in 13 µL of Elution Buffer (EB).

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
82. Incubate on at 37 °C for 10 min at Room temperature, agitate the sample for 10s every 2 min.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
83. Pellet beads on magnet until the eluate is clear and colorless.
SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
84. Remove and retain 13 µL of eluate into a clean 1.5ml low DNA binding tube.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
85. Quantify 1 µL of eluted sample using a Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit (recovery aim ~ 430 ng in total).

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
86. Make up the library to 12 µL at 10-20 fmol.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
87. Put the library on ice until ready to load or store the library at -20 °C for future sequencing.

SECTION: Priming and loading the SpotON Flow Cell
88. Check the number of pores in your flow cell.
 

SECTION: Priming and loading the SpotON Flow Cell
88.1. Turn on GridION (or MinION Mk1C) device. Make sure all the connections for the display, mouse, keyboard, and internet are ready.
SECTION: Priming and loading the SpotON Flow Cell
88.2. Depending on the number of pooled samples, get one to four new flow cells from the fridge and check the expiration date.
SECTION: Priming and loading the SpotON Flow Cell
88.3. Double-click the MinKNOW icon shown on the desktop to initiate the program.
SECTION: Priming and loading the SpotON Flow Cell
88.4. Use Oxford Nanopore Community username and password to login.
SECTION: Priming and loading the SpotON Flow Cell
88.5. Select the device shown on the screen.
SECTION: Priming and loading the SpotON Flow Cell
88.6. Open the lid of GridION (or MinION Mk1C) and insert the flow cells under the clips, press down the flow cell to ensure good thermal and electrical contact.
SECTION: Priming and loading the SpotON Flow Cell
88.7. The Sequencing Overview tab should show the flow cell not checked in each position in use.
SECTION: Priming and loading the SpotON Flow Cell
88.8. Navigate to the Start tab and select Flow Cell Check.
SECTION: Priming and loading the SpotON Flow Cell
88.9. Select the flow cells to assign the flow cell type FLO-MIN114 from the dropdown menu.
SECTION: Priming and loading the SpotON Flow Cell
88.10. Click Start to begin the flow cell check.
SECTION: Priming and loading the SpotON Flow Cell
88.11. Record the port number and date of checking on the original package of the flow cell. The flow cell with less than 800 pores should not be used for the sequencing. If the flow cell is not expired, contact Oxford Nanopore Company for customer service.
SECTION: Priming and loading the SpotON Flow Cell
88.12. If the flow cell is going to be used immediately, keep it on the GridION or MinION Mk1C sequencer for priming. Otherwise put the flow cell back to the original pouch, store at 4 °C for next day use. The opened flow cell should be used within one week.

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
89. 
BEFORE STARTING:
Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT) and one tube of Flow Cell Flush (FCF) at  Room temperature. Mix SB by tapping or pipetting (DO NOT Vortex) and vortex the other tubes. Spin down tubes at Room temperature.

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
91. Slide open the GridION lid (or MinION Mk1C) and insert flow cell with minimum 800 pores.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
92. Slide the priming port cover clockwise to open the priming port.
 

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
93. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume (20-30 μl) to remove any bubbles:
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
93.1. Set a P1000 pipette to 200 μl. Insert the tip into the priming port. Turn the volume adjustment wheel counter-clockwise until the dial shows 220-230 μl, or until you can see a small buffer volume entering the pipette tip.
 

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
94. Prepare the flow cell priming mix and prime flow cells.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
94.1. Using a 2.0 mL low DNA binding tube, prepare flow cell priming mix with components as follows, mix by inverting the tube and pipetting. 
     Component      Volume        Bovine Serum Albumin (BSA) (50 mg/ml)      5 μl        Flow Cell Tether (FCT)      30 μl        Flow Cell Flush (FCF)      1170 μl        Final total volume      1205 μl   
 
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
94.2. Load 800 µL of the priming mix into each flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 min.

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
95. Prepare the library for loading. 
 

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
95.1. Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
95.2. In a new tube, prepare each library for loading as follows: 
     Component      Volume        Sequencing Buffer (SB)      37.5 μl        Library Beads (LIB)      25.5 μl        DNA library      12 μl        Final total volume      75 μl   
 
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
96. Complete the flow cell priming.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
96.1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
96.2. Load 200 µL of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
97. Loading samples.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
97.1. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
97.2. Add 75 µL of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next drop.

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
97.3. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the GridION lid.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
98. 
Double-click the MinKNOW icon displayed on the desktop to initiate the program. 
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
99. Use Oxford Nanopore Community username and password to login or continue as Guest.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
100. Select the device shown on the screen.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
101. Go to the Start tab, and click the Start Sequencing option to choose the running parameters.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
102. Click Continue to Kit Selection to move to the next page.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
103. Click Continue to Run Options to choose run parameters.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
104.  Click Continue to Analysis to choose basecalling and Barcoding parameters.

SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
105. Click Continue to output to the next page.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
106. Click Continue to final review to proceed.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
107. Review the settings listed in the Run Setup page. Correct any errors. Select Start to run the experiment.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
108. The system will automatically navigate the Sequencing Overview when sequencing starts.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
109. 48 hrs later, check the sequencing data. Use 1 mL pipette to remove 1 mL waste solution in the waste channel via waste port 1 (see Appendix 1 under Guidelines & Warnings tab). Remove the flow cells on the device, put it back in the original package, and turn off the device.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19. Prepare MBD2-Fc Protein and Magnetic Beads in Microbiome DNA Enrichment Kit for host DNA depletion.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.1. Place one tube of 3 mL 5X Bind/wash buffer, NEBNext Protein A Magnetic Beads, and NEBNext MBD2-Fc Protein on ice.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.2. Add 12 mL Nuclease-free water into the 3 mL 5X Bind/wash buffer in 15mL tube to make 1X Bind/wash buffer and relabel the tube as 1X buffer.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.3. Resuspend NEBNext Protein A Magnetic Beads by gently pipetting the slurry up and down until the suspension is homogeneous; avoid bobbling. 
 

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.4. In one 1.5mL tube, add 8 µL of MBD2-Fc protein and 80 µL of Protein A Magnetic Beads. Mix by pipetting up and down (avoid bobbling) until the beads are completely homogeneous, at least 5-10 times. 
 

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.5. Mix the beads in a Hula mixer for 3 min at Room temperature.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.6. Spin the tube for 5 s and use pipetting to resuspend the bottom beads if any. If the mix is prepared for multiple samples, aliquot 80 µL mix to 1.5mL tubes.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.7. Briefly spin the tube and place in the magnetic rack for 2–5 minutes or until the beads have collected to the wall of the tube and the solution is clear.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.8. Carefully remove the supernatant with a pipette without disturbing the beads.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.9. Add 500 µL of 1X Bind/wash Buffer (kept on ice) to the tube to wash the beads. Pipette up and down until the beads are completely homogeneous, at least 5-10 times.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.10. Mix the beads in a Hula mixer for 3 min atRoom temperature.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.11. Briefly spin the tube and place on the magnetic rack for 2–5 minutes or until the beads have collected to the wall of the tube and the solution is clear.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.12. Carefully remove the supernatant with a pipette without disturbing the beads.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.13. Repeat steps 119.9 to 119.12.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.14. Remove the tube from the rack and add 80 µL of 1X Bind/wash Buffer (kept on ice) to resuspend the beads.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.15. The MBD2-Fc-bound magnetic beads now are ready to use for host DNA depletion and stable for up to 7 days at 4 °C.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19. Prepare riboPOOL Pan-Mammal Kit.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.1. Upon arrival, store the reagents in the kit as instructed on the bottles.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.2. When using the kit for the first time, centrifuge the riboPOOL probe tube (RP) at 11000 x g, 30 s before opening. Add 30 µL kit provided nuclease-free water and vortex well to resuspend the riboPOOL probe. Aliquot the 5 µLRP in 1.5ml tubes after resuspension. Store RP aliquots at -20 °C. Avoid freeze-thaw cycles.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19. Host rRNA depletion
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.1. Before starting, set up 2 programs on a thermocycler.
68 °C, 10 min, then slowly cool down to 37 °C at a speed 3 °C/min (0.05°C/sec); when reached, keep the temperature at 37 °C.
37 °C, 15 min, then 50 °C, 5 min.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.2. Transfer TNA containing around 500 ng RNA to a 200μL PCR tube, adjust volume with nuclease-free water to 13.7 µL. For example, if the RNA concentration of the TNA sample is 100 ng/ul, transfer 5 µL of the sample to a 200μL PCR tube and add 8.7 µL nuclease-free water. When the RNA concentration is lower than 36.5 ng/µl use 13.7 µL sample without volume adjustment.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.3. Probe Hybridization: add reagents as the following table for one sample. For processing multiple samples, make a master mix with a 10% overage.
 
     Component      Volume (μL)        TNA      13.7        RP      0.3        4X   Hybridization Buffer (HB)      5        RNaseOUT   (40U/ μL)      1        Total       20   
 
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.4. Mix by pipetting, then spin down briefly.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.5. Place the tube in the thermocycler and run the first program 68 °C cool down to 37 °C set at step 121.1.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.6. Prepare streptavidin-coated magnetic beads (SMB): Vortex SMB beads at medium speed. Transfer 45 µL bead suspension per sample to a new 1.5mL tube. For multiple sample processing, calculate the amount of SMB needed by multiple 45 with the sample number.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.7. Briefly spin down, place the tube on a magnetic rack and wait for 1 min.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.8. Aspirate and discard all supernatant.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.9. Add 40 µL Depletion Buffer per sample (i.e. 240 µL for 6 samples, 480 µL for 12 samples) and tap the tube to resuspend beads.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.10. Repeat steps 121.7 to 121.9. Beads are now resuspended in Depletion Buffer. 
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.11. Move the tube in the thermocycler at step 121.5 to a PCR tube rack. 
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.12. Add 40 µL beads from step 121.10 to the PCR tube and mix by pipetting.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.13. Put the tube back into the thermocycler for incubation at 37 °C for 15 min, then 55 °C for 5 min.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.14. After incubation, briefly spin down droplets.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.15. Transfer 60 µL sample-bead mix in the PCR tube to a new 1.5mL tube.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.16. Place the 1.5mL tube on the magnet rack for 2 min then carefully transfer the 60 µL supernatant to a new 1.5mL tube. The supernatant now contains host rRNA-depleted TNA. Keep the supernatant on ice for the next section of Host DNA depletion.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19. Host DNA depletion.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.1. Add 60 µL host rRNA depleted sample from the previous step (121.16) to a tube containing 80 μL MBD2-Fc-bound magnetic beads prepared at step 119.14. 
 


SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.2. Add 35 µL 5X Bind/wash buffer from the 1 mL aliquot prepared at step 118.1.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.3. Mix the sample in the Hula mixer for 15 min at Room temperature.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.4. Briefly spin the tube and place it on the magnetic rack for 5 min until the beads have collected to the wall of the tube and the solution is clear.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.5. Carefully transfer 175 µL supernatant with a pipette, without disturbing the beads to a clean 1.5mL microcentrifuge tube. The sample at this stage is host rRNA and DNA depleted in buffer. Store this sample at -20 °C or proceed directly to the sample cleanup described in the section A6 before processed to the SOP of blood testing.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19. AMPure XP Bead Cleanup.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.1. Vortex AMPure XP Beads to resuspend.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.2. Add 175 µL resuspended AMPure XP beads to the sample at step 122.5.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.3. Mix well by pipetting up and down at least 10 times. 
 

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.4. Incubate samples for at least 5 min at Room temperature.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.5. Quickly spin the sample, then place the tube on the magnet rack to separate the beads from the supernatant.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.6. After 2-5 minutes (or when the solution is clear), be careful not to disturb the beads, and remove and discard the supernatant.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.7. Add 400 µL of freshly prepared 80% ethanol to the tube on a magnetic rack. Incubate at Room temperature for 30 s, then carefully remove and discard the 80% ethanol without disturbing the beads.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.8. Repeat the previous step.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.9. Briefly spin the tube, place it back on the magnetic stand and remove traces of ethanol with a p10 pipette tip.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.10. Air dry the beads for 2 min while the tube is on the magnetic rack with the lid open.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.11. Adding 30 µL of nuclease-free water to the beads.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.12. Briefly spin the sample and mix well by pipetting up and down 10 times.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.13. Incubate for 2 min at Room temperature.

SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.14. Briefly spin the sample, then place the tube on the magnetic rack for 2-5 minutes (or when the solution is clear).
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
19.15. Transfer the supernatant to a new 1.5mL tube.
SECTION: APPENDIX 1. HOST DNA &amp; rRNA DEPLETION for TNA from WHOLE BLOOD &amp; BUFFY COAT
18.16. Use 1 µL for DNA/ RNA concentration measurement. Refer to REDI-NET SOP B-2 for the method of DNA/RNA quantification and storage. Recode the concentration on REDI-NET DCS B-4 Blood Testing.

SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
19. After Double-clicking the MinKNOW icon, the login page should show up. Use Oxford Nanopore Community username and password to login.
 

SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
20. Select the device shown on the screen.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
20. Go to Start tab, select the “Start Sequencing” option to choose the running parameters.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
21. Type in the (1) experiment name, (2) sample ID and (3) choose flow cell type from the drop-down menu. Use (4) “Select all available” to select all the connected flow cells or use the diagram above to select specific flow cells to run. (5) Select Continue to Kit Selection to move to the next page.
 

SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
22. elect the kit SQK-NBD114-96 from the Kit Selection menu. Select Continue to Run Options to choose run parameters.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
23. Select the speed: Accurate (260bp).
 
 
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
24. Set run length to 2880 min and Minimum read length 200 bp. Let adaptive sampling off. Select Continue to analysis for output setting.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
25. Turn on Basecalling and Barcoding. 
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
26. Set up Option of Basecalling model in High-accuracy basecalling. Save the setting.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
27. Set up Barcoding options. Turn on Trim barcodes and Mid-read barcode filtering. Save the settings and Continue to output.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
28. Select the output data location, format, and filtering options. Check the box for Raw reads and select POD5. Check the box FASTQ of Basecalled reads. Keep the filter score as the system default. Select “Continue to final review” to proceed.
 
SECTION: APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
29. Review the settings listed in the Run Setup page. Correct any errors. Select 'Start' to run the experiment. The system will automatically navigate the Sequencing Overview when sequencing starts. 
 
SECTION: cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
14. BEFORE START: Thaw 60 μM stock Random Primer Mix (NEB, S1330S) at Room temperature. DO NOT USE the Random Primer provided by the NEBNext First Strand Synthesis Module. Thaw Random Primer Mix solution, NEBNext First Strand Reaction Buffer, NEBNext Second Strand Reaction Buffer at Room temperature then place on ice. Vortex the vials briefly, spin done by centrifugation for 5 s, and place on ice. First and Second Strand Enzyme Mix are not frozen, should be briefly centrifuged and placed  on ice before use.

SECTION: SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
70. BEFORE STARTING: Thaw Short Fragment Buffer (SFB), Elution Buffer (EB), and NEBNext Quick Ligation Reaction Buffer (5×) at Room temperature, mix by vortexing, spin down, and place on ice. Check that the contents or each tube are clear of any precipitate. Spin down the T4 Ligase and the Native Adapter (NA), and place on ice.

SECTION: Priming and loading the SpotON Flow Cell: Flow cell priming
90. Check the air bubble of priming pore.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
101.1. Type in the Experiment Name using the scheme, [YYYY_MM_DD_Approach(gDNA or TNA)_Sample type (soil, water,… etc.)]
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
101.2. Type in Sample ID (same as experiment name)
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
101.3. Choose flow cell FLO-MIN114 from the drop-down menu
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
102.1. Click the kit SQK-NBD114-96 from the Kit Selection menu.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
103.1. Set run length to 2880 min and minimum read length 200 bp. Let adaptive sampling be unchecked.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
104.1. In the Basecalling options, checkup the basecalling with configuration:  High accuracy basecalling.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
104.2. In the Barcoding options, turn on the Trim barcodes and Mid-read barcoding filtering.
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
104.3. Do not turn on the Alignment option. 
SECTION: Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
105.1. Select the output data location, format, and filtering options. Check up the box for Raw reads in POD5 format and Basecalled reads in FASTQ format. Keep the filter score as the system default.