Oct 06, 2022

Public workspaceHIV WGS - 400bp Amplicon Tiling - Oxford Nanopore Technology Protocol V.1

DOI
dx.doi.org/10.17504/protocols.io.e6nvwj5p2lmk/v1
  • 1Association of Public health Laboratories, Oregon State Public Health Laboratory;
  • 2Association of Public Health Laboratories
  • Eugene Yeboah: Contributing authors: Laura Tsaknaridis, Vanda Makris, John Fontana, Shane Sevey;
Eugene Yeboah
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwj5p2lmk/v1
Protocol Citation: Eugene Yeboah, Noah C Hull 2022. HIV WGS - 400bp Amplicon Tiling - Oxford Nanopore Technology Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj5p2lmk/v1
Manuscript citation:
Freed, N. E., Vlková, M., Faisal, M. B., & Silander, O. K. (2020). Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding. Biology Methods and Protocols, 5(1), bpaa014. https://doi.org/10.1093/biomethods/bpaa014CrossRefPubMedGoogle Scholar
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 05, 2022
Last Modified: October 06, 2022
Protocol Integer ID: 70862
Keywords: HIV, Whole Genome Sequecing, WGS, NGS, ONT, Oxford Nanopore Technology,
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Abstract
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:
ABCDEF
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:
ABCDEF
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


Materials
EQUIPMENT / SUPPLIES / REAGENTS:

AB
- Diluent (DIL)
- Elution Buffer (EB)
- Flush Buffer (FB)
- Flush Tether (FLT)
- Loading Beads II (LBII)
- Loading Solution (LS)
- Rapid Adapter F (RAP-F)
- Rapid Barcode Plate (RB96)
- Sequencing Buffer II (SBII)
- SPRI beads (SPRI)
- Storage Buffer (S)
- Wash Mix (WMX)
Absolute Ethanol, 200 proof, Molecular Biology Grade Cat. # T038181000
Biohazard containers for sharps, assorted sizes
Coverage Spray HB Plus Steris #1624-77 or VWR #4212-963
Disposable powder-free gloves
Eppendorf® Centrifuge 5430/5430R Cat. # EP022620645
Eppendorf™ PCR Cooler Cat. # 05-403-00
EQUIPMENT
Flow Cell Wash Kit Nanopore Cat# EXP-WSH004
Gentle rotator mixer or HULA mixer
HIV-1/HIV-2 Primers IDT – Custom
Kim Wipes
Low Protein Binding Collection Tubes (1.5 ml) Cat. # 90410
LunaScript® RT SuperMix Kit NEB Cat# E3101
Magnetic Separator, suitable for 1.5ml tubes
MicroAmp™ 8-Cap Strip, clear Cat. # N8010535
MicroAmp™ Optical 8-Cap Strips Cat. # 4323032
MicroAmp™ Optical 96-Well Reaction Plate Cat. # N8010560
myFuge 12 Micro-Centrifuge Cat. # 50-550-338
ONT Sequencer
PCR Cabinet – NuAire, model 126-300 Cat. # A35422
Permanent marking pens
Q5® Hot Start High-Fidelity SX Master Mix NEB Cat# M0494
Qubit® 4.0 Fluorometer
Qubit™ Fluorometry 4.0 Cat. # Q33238
Qubit™ 1X dsDNA HS Assay Kit Cat. # Q33230 or Q33231
Rainin Multichannel Pipettors – 20ml and 200ml
Rainin Pipettors – 2ml, 20ml, 200ml and 1000ml
Rainin Repeater Pipettor – 20ml and 200ml
Rainin 1000ml filter tips, Terra rack Cat. # 17014967
Rainin 200ml filter tips, Terra rack Cat. # 17014963
Rainin 20ml filter tips, Terra rack Cat. # 17014961
Rapid Barcoding Kit: Nanopore Cat# SQK-RBK110.96
REAGENTS
RNase Away™ Fisher Scientific #21-236-21 or equiv.
SimpliAmp™ Thermal Cycler or similar Cat. # A35422
SpotON Flow Cell (R9.4.1) Nanopore Cat# FLO-MIN106D
SUPPLIES
UltraPure™ DNase/RNase-Free Distilled Water Cat. # 10977015
Vortex Mixer

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.
Place 96-well PCR plate on an Eppendorf® PCR cooler (stored at -20oC).
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.
Cover the plate and move the PCR plate containing master mix from the PCR Clean Room to the processing bench for sample addition.
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.
Seal the plate with MicroAmp™ 8-cap strips and briefly centrifuge to collect droplets.
Return the RT plate to the Eppendorf® PCR cooler until thermocycler reaches 25oC.
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

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.)
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
15. Place 96-well PCR plate on an Eppendorf® PCR cooler (stored at -20oC).
16. Using a repeater pipettor aliquot 22.5 μl of each master mix into the appropriate wells of the 96-well plate.
17. Cover and store at 4oC until reverse transcription protocol is complete.
18. Remove the RT plate from the thermocycler and centrifuge.
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.
21. Seal the plate with 8-cap strips and briefly centrifuge.
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

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

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.
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
26. Remove the cDNA plate from the thermocycler and centrifuge.
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.)
29. Gently mix by pipetting the contents of each well up and down. Total volume = 25 μl per well.
30. Seal the plate with 8-cap strips and briefly centrifuge.
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
ABCDE
REPS 1 45X Hold
TIME  0:30 0:15 5:00 ∞
TEMP  98oC 98oC 65oC 4oC

Addition of rapid barcodes
Thaw Rapid Barcode Plate and bring SPRI beads to room temperature ~ 1 hour before PCR is complete.
35. Spin down the Rapid Barcode Plate.
36. Remove the primer-specific cDNA plate from the thermocycler and centrifuge briefly to collect the contents at the bottom of the wells.
37. Place the plate on an Eppendorf® PCR cooler.
38. Carefully remove the 8-cap strips from the cDNA plate to prevent any splashing.
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.
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).
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.
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.
43. Seal the plate with 8-cap strips and briefly centrifuge.
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

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.
47. Carefully remove the 8-cap strips from the BAP plate to prevent any splashing.
48. Pool the barcoded samples into a 5 ml Eppendorf DNA LoBind tube. ~10 μl per sample
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
50. Resuspend the SPRI beads by vortexing.
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

53. Incubate at room temperature on a gentle rotator mixer (~700 rpm) for 5 minutes.
54. While incubating, prepare 3 ml of fresh 80% ethanol in nuclease-free water (2400 μl ethanol 600 μl water).
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.
56. When the solution clears, keep the tube on the magnet and pipette off the supernatant and discard.
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.
58. Repeat the previous step.
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.
60. Remove the tube from the magnet and resuspend the pellet in 30 μl Elution Buffer (EB). Gently mix by pipetting up and down.
61. Incubate for 10 minutes at room temperature.
62. Place the tube on the magnet until the eluate is clear and colorless.
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.
64. Proceed to quantifying the DNA concentration using the Qubit dsDNA HS Assy Kit. (Or any preferred quantification method)
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: .
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.
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.
70. On the Qubit™ 4.0 Fluorometer home screen, select dsDNA then 1x dsDNA High Sensitivity.
71. Measure the prepared standards.
72. Select the sample concentration as “ng/μl” with an input value of 1 μl.
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.
76. Add 1 μl of Rapid Adapter F (RAP F) to the 11 μl normalized barcoded DNA and mix gently.
77. Incubate at room temperature for 5 minutes.
78. After incubation, place the prepared library in a cold rack until ready to load onto the SpotON flowcell.
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.
81. Mix the SBII, FB and FLT tubes by vortexing. Spin down the SBII and FLT tubes.
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.
83. QC the SpotON flowcell using the MinKNOW software before proceeding. QC passes if total active pores are >800.
84. After QC is complete, move the unit including the flowcell to the bench for priming and loading.
85. Slide the priming port cover clockwise to open the priming port.
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.
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)
89. Set a P1000 to 200 μl
90. Insert the tip into the priming port
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.
92. Visually inspect that there is continuous buffer from the priming port across the sensor array.
93. Load 800 ul of the priming mix into the flow cell via the priming port without introducing bubbles. Wait 5 minutes.
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
Complete the flow cell priming
Gently lift the SpotON sample port cover to make the sample port accessible.
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.
Immediately, mix the prepared library by pipetting up and down gently.
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.
Replace the SpotON sample port cover, making sure it is seated correctly, close the priming port, and close the sequencer lid. Proceed to sequencing