TITLE: HIV WGS - 400bp Amplicon Tiling - Oxford Nanopore Technology Protocol
AUTHORS: Eugene Yeboah, Noah C Hull

[DESCRIPTION]
OSPHL in collaboration with APHL, will evaluate the performance of the Oxford Nanopore Technology sequencers for HIV genome sequencing using a modified protocol of the ARTIC amplicon v3:

Nanopore Protocol PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96) Library preparation Version: PCTR_9125_v110_revE_24Mar2021

The modifications include: 
Switching to the Nanopore RAPID barcoding, which requires less time (10 min) and fewer reagents. 
HIV Primer sequences designed by Primal Scheme.
HIV-1:
https://www.dropbox.com/sh/xnhpektoen7chdd/AAAT789gnEnwbbCjF_EMuRu8a?dl=0
HIV-2: 
https://www.dropbox.com/sh/2sco4ffr30ids7a/AAD0z-VXSXElamsuys7zsmpLa?dl=0

: Primer Sequences

HIV-1:
 
 
 
 
 
 
     name      pool      seq      size      %gc      tm (use 65)        HIV-1_v1.0_1_LEFT      1      TGGTTAGACCAGATCTGAGCCT      22      50      60.48        HIV-1_v1.0_1_RIGHT      1      TTTCTTTCCCCCTGGCCTTAAC      22      50      60.68        HIV-1_v1.0_3_LEFT      1      GCTTTAGACAAGATAGAGGAAGAGCA      26      42.31      60.85        HIV-1_v1.0_3_RIGHT      1      TTCCTGCTATGTCACTTCCCCT      22      50      61.02        HIV-1_v1.0_5_LEFT      1      TTGGATGACAGAAACCTTGTTGG      23      43.48      59.62        HIV-1_v1.0_5_RIGHT      1      AAGAAAATTCCCTGGCCTTCCC      22      50      61.01        HIV-1_v1.0_7_LEFT      1      ATTAGAAGAAATGAGTTTGCCAGGAA      26      34.62      59.72        HIV-1_v1.0_7_RIGHT      1      TTCTTTATGGCAAATACTGGAGTATTGT      28      32.14      59.98        HIV-1_v1.0_9_LEFT      1      GAGACACCAGGGATTAGATATCAGT      25      44      59.61        HIV-1_v1.0_9_RIGHT      1      CCCTGGGTAAATCTGACTTGCC      22      54.55      61.13        HIV-1_v1.0_11_LEFT      1      AGAGCCATTTAAAAATCTGAAAACAGGA      28      32.14      60.77        HIV-1_v1.0_11_RIGHT      1      CAGTCTTCTGATTTGTTGTGTCAGT      25      40      60.08        HIV-1_v1.0_13_LEFT      1      GTCAGTGCTGGAATCAGGAAAGT      23      47.83      61.06        HIV-1_v1.0_13_RIGHT      1      CGTAGCACCGGTGAAATTGCT      21      52.38      61.64        HIV-1_v1.0_15_LEFT      1      AGACATAATAGCAACAGACATACAAACT      28      32.14      59.77        HIV-1_v1.0_15_RIGHT      1      CCAATCTAGCATCCCCTAGTGG      22      54.55      59.88        HIV-1_v1.0_17_LEFT      1      CAAGCAGGACATAACAAGGTAGGA      24      45.83      60.65        HIV-1_v1.0_17_RIGHT      1      TCCAGGGCTCTAGTCTAGGATC      22      54.55      59.81        HIV-1_v1.0_19_LEFT      1      TCTCTATCAAAGCAGTAAGTAGTACATGT      29      34.48      60.52        HIV-1_v1.0_19_RIGHT      1      GCATGTGTGGCCCAAACATTAT      22      45.45      60.28        HIV-1_v1.0_21_LEFT      1      AGCGGGAGAATGATAATGGAGAA      23      43.48      59.61        HIV-1_v1.0_21_RIGHT      1      GCATTGTCCGTGAAATTGACAGA      23      43.48      60.31        HIV-1_v1.0_23_LEFT      1      AGCTAGCAAATTAAGAGAACAATTTGGA      28      32.14      60.61        HIV-1_v1.0_23_RIGHT      1      TTCACTTCTCCAATTGTCCCTCA      23      43.48      59.86        HIV-1_v1.0_25_LEFT      1      CTATTGAGGCGCAACAGCATCT      22      50      61.5        HIV-1_v1.0_25_RIGHT      1      ACCTACCAAGCCTCCTACTATCA      23      47.83      60.05        HIV-1_v1.0_27_LEFT      1      ACCACCGCTTGAGAGACTTACT      22      50      61.27        HIV-1_v1.0_27_RIGHT      1      TGCTCCATGTTTTTCCAGGTCT      22      45.45      60.34        HIV-1_v1.0_29_LEFT      1      CACACACAAGGCTACTTCCCTG      22      54.55      61.31        HIV-1_v1.0_29_RIGHT      1      AACCAGAGAGACCCAGTACAGG      22      54.55      61.01        HIV-1_v1.0_2_LEFT      2      TAGAAGGAGAGAGATGGGTGCG      22      54.55      61.19        HIV-1_v1.0_2_RIGHT      2      TTTTGGCTGACCTGATTGCTGT      22      45.45      61.2        HIV-1_v1.0_4_LEFT      2      GCTGCAGAATGGGATAGAGTGC      22      54.55      61.56        HIV-1_v1.0_4_RIGHT      2      TTCTTCTAGTGTAGCCGCTGGT      22      50      61.33        HIV-1_v1.0_6_LEFT      2      GGAAGGACACCAAATGAAAGATTGT      25      40      60.37        HIV-1_v1.0_6_RIGHT      2      TGTCCACAGATTTCTATGAGTATCTGA      27      37.04      59.94        HIV-1_v1.0_8_LEFT      2      AGTAGAAATTTGTACAGAGATGGAAAAGG      29      34.48      60.47        HIV-1_v1.0_8_RIGHT      2      AAGGCTCTAAGATTTTTGTCATGCT      25      36      59.67        HIV-1_v1.0_10_LEFT      2      CAGCCTATAGTGCTGCCAGAAA      22      50      60.6        HIV-1_v1.0_10_RIGHT      2      TTTGCACTGCCTCTGTTAATTGT      23      39.13      59.62        HIV-1_v1.0_12_LEFT      2      GGGAGACTAAATTAGGAAAAGCAGGA      26      42.31      61.02        HIV-1_v1.0_12_RIGHT      2      AGCCATTGCTCTCCAATTACTGT      23      43.48      60.57        HIV-1_v1.0_14_LEFT      2      GGGCAGGAAACAGCATATTTTCT      23      43.48      59.81        HIV-1_v1.0_14_RIGHT      2      TGCTGTCCCTGTAATAAACCCG      22      50      60.54        HIV-1_v1.0_16_LEFT      2      GGGAAAGCTAGGGGATGGTTTT      22      50      60.48        HIV-1_v1.0_16_RIGHT      2      TCGTAACACTAGGCAAAGGTGG      22      50      60.47        HIV-1_v1.0_18_LEFT      2      GCAACAACTGCTGTTTATCCATTTT      25      36      59.91        HIV-1_v1.0_18_RIGHT      2      TTTCCTATATTCTATGATTACTATGGACCAC      31      32.26      59.61        HIV-1_v1.0_20_LEFT      2      TACCTGTGTGGAAGGAAGCAAC      22      50      60.67        HIV-1_v1.0_20_RIGHT      2      TGCATATTCTTTCTGCACCTTACCT      25      40      60.96        HIV-1_v1.0_22_LEFT      2      GCCAGTAGTATCAACTCAACTGCT      24      45.83      60.94        HIV-1_v1.0_22_RIGHT      2      ACAGTAGAAAAATTCCCCTCCACA      24      41.67      60.22        HIV-1_v1.0_24_LEFT      2      GGGCTGCTATTAACAAGAGATGGT      24      45.83      61.01        HIV-1_v1.0_24_RIGHT      2      AGGTATCTTTCCACAGCCAGGA      22      50      61.02        HIV-1_v1.0_26_LEFT      2      TGGGCAAGTTTGTGGAATTGGT      22      45.45      61.41        HIV-1_v1.0_26_RIGHT      2      ACCAATATTTGAGGGCTTCCCAC      23      47.83      61.14        HIV-1_v1.0_28_LEFT      2      TGGATGGCCTACTGTAAGGGAA      22      50      60.75        HIV-1_v1.0_28_RIGHT      2      AGCTTGTAGCACCATCCAAAGG      22      50      61.06   

HIV-2:
     name      pool      seq      size      %gc      tm (use 65)        HIV-2_v1.0_1_LEFT      1      TGCAAGGGATGTTTTACAGTAGGA      24      41.67      60.28        HIV-2_v1.0_1_RIGHT      1      CCAAGTATGGTTGTTCCTGTTATTCA      26      38.46      60.01        HIV-2_v1.0_3_LEFT      1      GCATTGTATTCAGTCGCTCTGC      22      50      60.46        HIV-2_v1.0_3_RIGHT      1      ACTCCGTCGTGGTTTGTTCCT      21      52.38      62.01        HIV-2_v1.0_5_LEFT      1      AAAACATATTGTGTGGGCAGCG      22      45.45      60.53        HIV-2_v1.0_5_RIGHT      1      TCCTCCACTAATTTTACCCATGCAT      25      40      60.73        HIV-2_v1.0_7_LEFT      1      ATTCGCAGCACCCAATACCAG      21      52.38      61.12        HIV-2_v1.0_7_RIGHT      1      GCGGTTAGCATCTCTTCTAGGG      22      54.55      60.47        HIV-2_v1.0_9_LEFT      1      CAGGACACATCATGGCAAACTG      22      50      60.27        HIV-2_v1.0_9_RIGHT      1      TTGTCCCCTAATTCTATTCCTGCT      24      41.67      59.6        HIV-2_v1.0_11_LEFT      1      GGAAAGATGGACCAAGGCTGAA      22      50      60.74        HIV-2_v1.0_11_RIGHT      1      CTTCCATCCTTGTGGCAAGACT      22      50      60.74        HIV-2_v1.0_13_LEFT      1      GGGTTTTCTACTCCAGATGAGAAGT      25      44      60.43        HIV-2_v1.0_13_RIGHT      1      ACTTTTAGGATTTTCTCTTCCTGGTGT      27      37.04      61.14        HIV-2_v1.0_15_LEFT      1      GGTTAACATTTAACCTAGTAGGAGATCCT      29      37.93      60.78        HIV-2_v1.0_15_RIGHT      1      ACTTGTCTGATGCCTTGACTTACT      24      41.67      60.28        HIV-2_v1.0_17_LEFT      1      TCATTATAGTAGCAGTACATGTTGCAA      27      33.33      59.51        HIV-2_v1.0_17_RIGHT      1      CATGTTGATTAGTCTTTCTGCTGGG      25      44      60.54        HIV-2_v1.0_19_LEFT      1      ACTGGATAGTAGTCCCCACCTG      22      54.55      60.55        HIV-2_v1.0_19_RIGHT      1      ATGAGCTTGGGGATAGTTGCAG      22      50      60.61        HIV-2_v1.0_21_LEFT      1      GGGATGTCAGCAAGCTACACAA      22      50      61.06        HIV-2_v1.0_21_RIGHT      1      GGTTACATCCCGCTCTGAAGTG      22      54.55      61.17        HIV-2_v1.0_23_LEFT      1      TGGTAGGAATCAGCTGTTTGTTGT      24      41.67      61.01        HIV-2_v1.0_23_RIGHT      1      ATGTGGGTGGTCTCGAGGTT      20      55      60.85        HIV-2_v1.0_25_LEFT      1      AGCACTATTGGGATGCTATGAGG      23      47.83      60.25        HIV-2_v1.0_25_RIGHT      1      CTTCCCTCCATCTGCCTCCAAA      22      54.55      62.21        HIV-2_v1.0_27_LEFT      1      AGCAGATAATTAATACCTGGCATAAAGT      28      32.14      59.66        HIV-2_v1.0_27_RIGHT      1      CTGTTGCTGTTGCTGCACTATC      22      50      60.59        HIV-2_v1.0_29_LEFT      1      AATATGACATGGCAGGAGTGGG      22      50      60.34        HIV-2_v1.0_29_RIGHT      1      CAAGGCCACAAGTCGTAACCA      21      52.38      60.91        HIV-2_v1.0_31_LEFT      1      ATTCGCGAGGACTACGAGAGAG      22      54.55      61.55        HIV-2_v1.0_31_RIGHT      1      CCCTTCCTCTTTTTCTAGGTATATGTCT      28      39.29      60.51        HIV-2_v1.0_33_LEFT      1      ACTACAAGGCCTTCACTCTGTAC      23      47.83      60        HIV-2_v1.0_33_RIGHT      1      AGTACCGGCCAAGTACTGGT      20      55      60.56        HIV-2_v1.0_2_LEFT      2      GAGTTTGGGCACAAGTCAGGAT      22      50      61        HIV-2_v1.0_2_RIGHT      2      ACTGGCAGCTTTATTGAAGAGGT      23      43.48      60.5        HIV-2_v1.0_4_LEFT      2      CGAACAGGGACTTGAAGAGGAC      22      54.55      60.79        HIV-2_v1.0_4_RIGHT      2      TGGCAACCTTCTTTTGACTCCA      22      45.45      60.54        HIV-2_v1.0_6_LEFT      2      AAAAGAGGAAACTACCCCGTGC      22      50      60.99        HIV-2_v1.0_6_RIGHT      2      TCTGTAGATGTTCCCTACCGGT      22      50      60.21        HIV-2_v1.0_8_LEFT      2      TGGATGACCCAAACGCTGCTA      21      52.38      62.16        HIV-2_v1.0_8_RIGHT      2      GGGAAGTTGCGAGGCTTCTTTC      22      54.55      62.2        HIV-2_v1.0_10_LEFT      2      GTCACAGCGTACATCGAGGATC      22      54.55      61.03        HIV-2_v1.0_10_RIGHT      2      AGCTGGCCCTCTTTTTCCATTT      22      45.45      60.95        HIV-2_v1.0_12_LEFT      2      CTGCATTTACCCTACCAGCAGT      22      50      60.54        HIV-2_v1.0_12_RIGHT      2      GCTTTTGGATGTCATTGACTGTCC      24      45.83      60.93        HIV-2_v1.0_14_LEFT      2      ACCAAGAAGAAAAAGAATTAGAGGCA      26      34.62      59.67        HIV-2_v1.0_14_RIGHT      2      TCCTGCTTTTCCCTCTTTTGACT      23      43.48      60.38        HIV-2_v1.0_16_LEFT      2      AGCAGTCTATGTTGCATGGGTC      22      50      60.86        HIV-2_v1.0_16_RIGHT      2      GAAGAGTGCTGTCTGCCTTCCT      22      54.55      62.44        HIV-2_v1.0_18_LEFT      2      ACAATAGAAACAATAGTACTGATGGCAG      28      35.71      60.29        HIV-2_v1.0_18_RIGHT      2      CCTCCTCTAGGTCTTTTGTTCTGT      24      45.83      60.04        HIV-2_v1.0_20_LEFT      2      TCTTGCTTTACGGCAGGTGAAG      22      50      61.31        HIV-2_v1.0_20_RIGHT      2      GCAAGTGCACCCTCTCTTGAAA      22      50      61.51        HIV-2_v1.0_22_LEFT      2      GACATGGAGACACCCTTGAAGG      22      54.55      60.8        HIV-2_v1.0_22_RIGHT      2      CACGCGGGTATGCCATAGAAAA      22      50      61.5        HIV-2_v1.0_24_LEFT      2      TGGCAATGAATTGTAGCAGGGT      22      45.45      60.74        HIV-2_v1.0_24_RIGHT      2      AGCCTGAATAGTTGGTATCATTACATCT      28      35.71      60.77        HIV-2_v1.0_26_LEFT      2      TGTCAGGATTAGTGTTTCACTCTCA      25      40      60.08        HIV-2_v1.0_26_RIGHT      2      GCTGGTTACTGTTGATTCACAGG      23      47.83      60.06        HIV-2_v1.0_28_LEFT      2      CGTGCTAGGGTTCTTGGGTTTT      22      50      61.26        HIV-2_v1.0_28_RIGHT      2      TTCTTGTTGAATTTGGGCTTCTTCT      25      36      60.02        HIV-2_v1.0_30_LEFT      2      CCCGGTTATCTCCAACAGATCC      22      54.55      60.41        HIV-2_v1.0_30_RIGHT      2      CTTGGAACTGCGAGTATTCCCC      22      54.55      61.18        HIV-2_v1.0_32_LEFT      2      TCACATTTTATAAAAGAAAAAGGGGGACT      29      31.03      60.58        HIV-2_v1.0_32_RIGHT      2      CCCTCTTGCTTTCAGTTTTGCC      22      50      60.73        HIV-2_v1.0_34_LEFT      2      ACTTTCCAGAAGGGGCTGTAAC      22      50      60.41        HIV-2_v1.0_34_RIGHT      2      TGCTAGGGATTTTCCTGCCTTG      22      50      60.81   


 
 
 
 
 
 

[STEPS]
1. Reverse Transcription
The LunaScript® RT SuperMix Kit will be used to reverse transcribe HIV RNA for preparation to perform the Primer Pool PCR assay. NOTE: LunaScript® RT SuperMix will be kept in the PCR Clean room.
2. Place 96-well PCR plate on an Eppendorf® PCR cooler (stored at -20oC).   
3. Using a repeater pipettor aliquot 4 μl of LunaScript® RT SuperMix into each sample well of a 96‑well PCR plate according to plate map.   
4. Cover the plate and move the PCR plate containing master mix from the PCR Clean Room to the processing bench for sample addition.   
5. Add 16 μl of RNA extract to the appropriate sample well and gently mix by pipetting up and down. If less than 16 μl is available, make up the volume with nuclease-free water. Total volume = 20 μl per well.   
6. Seal the plate with MicroAmp™ 8-cap strips and briefly centrifuge to collect droplets.   
7. Return the RT plate to the Eppendorf® PCR cooler until thermocycler reaches 25oC.   
8. 10.  Load the plate into the SimpliAmp™ thermocycler, then run the “HIV REVERSE TRANSCRIPTION” method with the following parameters:  Set heated lid to 105oC  / Runtime ~14 minutes 

 
 
     REPS      1      1      1      Hold        TIME      2:00      10:00      1:00      ∞        TEMP      25oC      55oC      95oC      4oC   

 
 
9. PCR Primer Pool Preparation
Combine the following components to prepare the master mix for each primer pool (A or B). (Allow for pipetting loss by adding ~ 4 reactions per 32 samples.)   
10. PCR Primer Pool Preparation
Combine the following components to prepare the master mix for each primer pool (A or B). (Allow for pipetting loss by adding ~ 4 reactions per 32 samples.)  

NOTE: Use the HIV-1 RT and Primer Pool Preparation Worksheet to calculate the required amount of master mix. This can be made at the same time as the LunaScript® SuperMix RT plate and stored at 4oC until use.  
@10μM
Component                                            Volume
Nuclease-free Water                             8.90 μl
HIV-1 Primer Pool (A or B)                   1.10 μl
Q5® Hot Start HF 2X Master Mix       12.5 μl
Total volume per sample                      22.5 μl
11. 15.  Place 96-well PCR plate on an Eppendorf® PCR cooler (stored at -20oC).   
12. 16.  Using a repeater pipettor aliquot 22.5 μl of each master mix into the appropriate wells of the 96-well plate.   
13. 17.  Cover and store at 4oC until reverse transcription protocol is complete.   
14. 18.  Remove the RT plate from the thermocycler and centrifuge.   
15. 19.  Carefully remove the 8-cap strips and using a multi-channel pipettor transfer 2.5 μl of the RT reactions into the appropriate wells of the PCR plate containing the primer pools. Gently mix by pipetting the contents of each well up and down. Total volume = 25 μl per well.   

NOTE: It is useful to separate the plate in half with primer pool A in columns 1-6 and primer pool B in columns 7-12.  
16. 21.  Seal the plate with 8-cap strips and briefly centrifuge.   
17. 22.  Load the plate into the SimpliAmp™ thermocycler, then run the “HIV PCR PRIMERS” method with the following parameters:  Set heated lid to 105oC   /   Runtime ~5 Hours   

 
 
     REPS      1      45X      Hold        TIME        0:30      0:15      5:00      ∞        TEMP        98oC      98oC      65oC      4oC   

 
 
18. 24.  Repeat the same primer pool preparation from above (Step 1) and aliquot it into the same wells as in Step 1 into a new PCR plate in the same wells as the previous plate.   
19. 25.  NOTE: Use the HIV-1 RT and Primer Pool Preparation Worksheet to calculate the required amount of master mix.    
@10μM
Component                                                    Volume
Nuclease-free Water                                         8.90 μl
HIV-1 Primer Pool (A or B)                               1.10 μl
Q5® Hot Start HF 2X Master Mix                   12.5 μl
Total volume per sample                                  22.5 μl
20. 26.  Remove the cDNA plate from the thermocycler and centrifuge.   
21. 27.  Carefully remove the 8-cap strips and using a multi-channel pipettor transfer 2.5 μl of the 1st cDNA products into the corresponding wells of the new PCR plate containing the 2nd set of primer pools.   
(2.5 μl of 1st Pool A or B goes into corresponding 2nd Pool A or B of new plate.) 
22. 29.  Gently mix by pipetting the contents of each well up and down. Total volume = 25 μl per well.   
23. 30.  Seal the plate with 8-cap strips and briefly centrifuge.   
24. 31.  Load the plate into the SimpliAmp™ thermocycler, then run the “HIV PCR PRIMERS” method again. Same parameters as above.       
Set heated lid to 105oC   /   Runtime ~5 Hours 
 
 
     REPS      1      45X      Hold        TIME        0:30      0:15      5:00      ∞        TEMP        98oC      98oC      65oC      4oC   

 
 
25. Addition of rapid barcodes
Thaw Rapid Barcode Plate and bring SPRI beads to room temperature ~ 1 hour before PCR is complete.   
26. 35.  Spin down the Rapid Barcode Plate.   
27. 36.  Remove the primer-specific cDNA plate from the thermocycler and centrifuge briefly to collect the contents at the bottom of the wells.   
28. 37.  Place the plate on an Eppendorf® PCR cooler.   
29. 38.  Carefully remove the 8-cap strips from the cDNA plate to prevent any splashing.   
30. 39.  Place a clean 96-well PCR plate on a separate Eppendorf® PCR cooler (stored at -20oC) and combine each 25 μl cDNA reaction into a single well in the plate. Mix by pipetting up and down and set aside. Total volume per sample = 50 μl.   
31. 40.  Place another clean PCR 96-well sample plate on a separate Eppendorf® PCR cooler and using a repeater pipettor aliquot 2.5 μl of nuclease-free water into corresponding sample wells.  This is the Barcode Attachment Plate (BAP).   
32. 41.  Using a multichannel pipette, transfer 5 μl of the pooled cDNA products to the corresponding well of the Barcode Attachment Plate (BAP) and mix by pipetting.   
33. 42.  Using a multichannel pipette, transfer 2.5 μl of the appropriate Rapid Barcodes to the corresponding well of the Barcode Attachment Plate (BAP) and mix by pipetting. Be careful not to cross-contaminate the different wells. Total volume per sample = 10 μl.   
34. 43.  Seal the plate with 8-cap strips and briefly centrifuge.   
35. 44.  Incubate the plate on the thermocycler using the “RAPID BARCODE ATTACHMENT” protocol with the following parameters:     

 
 
     REPS      1      1        TIME        2:00      2:00        TEMP        30oC      80oC   

 
 
36. Pooling Samples and Clean-up
Remove the Barcode Attachment Plate (BAP) from the thermocycler and centrifuge briefly to collect the contents at the bottom of the wells.   
37. 47.  Carefully remove the 8-cap strips from the BAP plate to prevent any splashing.   
38. 48.  Pool the barcoded samples into a 5 ml Eppendorf DNA LoBind tube. ~10 μl per sample
39. Pool the barcoded samples into a 5 ml Eppendorf DNA LoBind tube. ~10 μl per sample
For example:    
 
     # Samples      24      48      96        Total Volume      ~240 μl      ~480 μl      ~960 μl   
 
40. 50.  Resuspend the SPRI beads by vortexing.   
41. Add an equal volume of resuspended SPRI beads to the pooled samples and mix by flicking the tube.
For example:     

 
 
     # Samples      24      48      96        Volume of SRPI   beads      ~240 μl      ~480 μl      ~960 μl   

 
 
42. 53.  Incubate at room temperature on a gentle rotator mixer (~700 rpm) for 5 minutes.   
43. 54.  While incubating, prepare 3 ml of fresh 80% ethanol in nuclease-free water (2400 μl ethanol 600 μl water).   
44. 55.  Spin down the sample tube briefly to bring any liquid down from the cap and place it on the magnet. A pellet will form on the side wall of the tube.   
45. 56.  When the solution clears, keep the tube on the magnet and pipette off the supernatant and discard.   
46. 57.  Without disturbing the beads, add 1.5 ml of the freshly prepared 80% ethanol. Rotate the tube on the magnet to move the beads front to back a few times to wash beads. Remove the ethanol using a pipette and discard.   
47. 58.  Repeat the previous step.   
48. 59.  Briefly spin down the tube and place it back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 seconds. Do not over dry the pellet to the point of cracking.   
49. 60.  Remove the tube from the magnet and resuspend the pellet in 30 μl Elution Buffer (EB). Gently mix by pipetting up and down.   
50. 61.  Incubate for 10 minutes at room temperature.   
51. 62.  Place the tube on the magnet until the eluate is clear and colorless.   
52. 63.  Remove and transfer the 30 μl eluate (containing the DNA library) into a clean 1.5 ml Eppendorf DNA LoBind tube. Dispose of the pelleted beads. Be careful not to transfer any of the beads.   
53. 64.  Proceed to quantifying the DNA concentration using the Qubit dsDNA HS Assy Kit. (Or any preferred quantification method)   
54. Library quantification and normalization
Analyze 1 μl of the amplified library using the Qubit™ 4.0 Fluorometer and the Qubit™ dsDNA HS Assay Kit. For more information, see the Qubit™ dsDNA HS Assay Kits User Guide

Determine the amplified library concentration using Qubit™ 4.0 Fluorometer: .   
55. 68.  Prepare the Qubit™ standards as directed in the user guide (10 μl standard  + 190 μl Qubit™ dsDNA HS Buffer ), mix well, and incubate for at least 2 minutes.   
56. 69.  For each sample, combine 1 μl of the amplified library with 199 μl of Qubit™ dsDNA HS Buffer, mix well, and incubate for at least 2 minutes.   
57. 70.  On the Qubit™ 4.0 Fluorometer home screen, select dsDNA then 1x dsDNA High Sensitivity.   
58. 71.  Measure the prepared standards.   
59. 72.  Select the sample concentration as “ng/μl” with an input value of 1 μl.   
60. 73.  Measure and record the library concentration.   

A total volume of 11 μl is required for the following step.

If concentration of the total pool is less than 600ng/μl, use library pool undiluted.
61. 76.  Add 1 μl of Rapid Adapter F (RAP F) to the 11 μl normalized barcoded DNA and mix gently.   
62. 77.  Incubate at room temperature for 5 minutes.   
63. 78.  After incubation, place the prepared library in a cold rack until ready to load onto the SpotON flowcell.   
64. Priming and Loading the SpotON Flow Cell
Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and Flush Buffer (FB) at room temperature.   
65. 81.  Mix the SBII, FB and FLT tubes by vortexing. Spin down the SBII and FLT tubes.   
66. 82.  Open the ONT sequencer lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.   
67. 83.  QC the SpotON flowcell using the MinKNOW software before proceeding. QC passes if total active pores are >800.   
68. 84.  After QC is complete, move the unit including the flowcell to the bench for priming and loading.   
69. 85.  Slide the priming port cover clockwise to open the priming port.   
70. Prime and load the SpotON Flow Cell
 In a 1.5 ml Eppendorf LoBind tube, prepare the flow cell priming mix by adding 30 μl of Flush Tether (FLT) to 1.17 ml of Flush Buffer (FB) and vortex to mix.   
71. 88.  Open the priming port and check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl)   
72. 89.  Set a P1000 to 200 μl   
73. 90.  Insert the tip into the priming port   
74. 91.  Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip.   
75. 92.  Visually inspect that there is continuous buffer from the priming port across the sensor array.
76. 93.  Load 800 ul of the priming mix into the flow cell via the priming port without introducing bubbles. Wait 5 minutes.   
77. Prepare the library for loading
Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting up and down. The LBII tube contains a suspension of beads that settle very quickly. It is vital that they are mixed immediately before use.   

In a new tube, prepare the library for loading as follows:

Reagent                                                    Volume
Sequencing Buff II (SBII)                         37.5 μl
Loading Beads II                                       22.5 μl
DNA Library                                               12 μl
Total volume                                              75 μl
78. Complete the flow cell priming
Gently lift the SpotON sample port cover to make the sample port accessible.   
79. Slowly load 200 ul of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the instruction of bubbles.   
80.   Immediately, mix the prepared library by pipetting up and down gently.   
81. Add 75 μl of sample to the flow cell via the SpotON port in a dropwise manner. Ensure each drop flows into the port before adding the next.   
82.   Replace the SpotON sample port cover, making sure it is seated correctly, close the priming port, and close the sequencer lid.   Proceed to sequencing