Jul 12, 2022

Public workspaceMonkeypox virus whole genome sequencing using combination of NextGenPCR and Oxford Nanopore V.1

This protocol is a draft, published without a DOI.
  • 1AmsterdamUMC location AMC, Dept. of Medical Microbiology & Infection Prevention, Amsterdam, The Netherlands;
  • 2Molecular Biology Systems B.V., Goes, The Netherlands
Anton van den Ouden
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Protocol Citation: Matthijs Welkers, M. Jonges, Anton van den Ouden 2022. Monkeypox virus whole genome sequencing using combination of NextGenPCR and Oxford Nanopore. protocols.io https://protocols.io/view/monkeypox-virus-whole-genome-sequencing-using-comb-ccc7sszn
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: In development
We are still developing and optimizing this protocol
Created: June 30, 2022
Last Modified: July 12, 2022
Protocol Integer ID: 65663
Keywords: Monkeypox virus, amplicon-based tiling PCR, Nanopore sequencing, Oxford Nanopore sequencing, Monkeypox
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Abstract
Rapid genomic surveillance of monkeypox virus (MPXV) can provide valuable insights in order to guide public health interventions. Current sequencing protocols make use of direct Oxford Nanopore Sequencing. However, the obtained depth is a limiting factor which prevents multiplexing samples on a flowcell making sequencing very costly. Here, we provide the protocol for a PCR-based amplicon tiling approach (inspired by SARS-CoV-2 Midnight Protocol by Nikki Freed et. al. and the ARTIC network) for MPXV consisting of a total of 88 primer sets divided over 2 amplicon pools. The amplicon size is ~2,5kB. Our approach will increase the coverage (depth) significantly and allow for multiplexing up to 20 samples on a single Nanopore flowcell.

In our experience clinical samples can be successfully sequenced with CT-values <25. Homopolymer regions will remain an issue in our approach, requiring manual curation of obtained consensus sequence genomes.
Primer pool preparation
Primer pool preparation
If required, resuspend lyophilised primers to a concentration of 100µM each
Note
Primers for this protocol were designed by Martin Schou Pedersen (Department of Clinical Microbiology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark) using Primal Scheme to generate overlapping 2500bp amplicons.
Primers are available as two pre-mixed pools from Monkeypox - MBS - Ultra Fast NEXTGENPCR®



Primers used to generate 2500 bp amplicons are here:
namepoolseqsize%gctm
monkeypox-2500_1_LEFT1AAAAATGTGTGACCCACGACCG225062,07
monkeypox-2500_1_RIGHT1CCGGGAACTTACGCTTTCAGAT225060,86
monkeypox-2500_3_LEFT1GTTAACGATGCGCACAATCTCG225061,01
monkeypox-2500_3_RIGHT1TAGTGAGAGCGAGAGTGACAGT225060,47
monkeypox-2500_5_LEFT1AATAGTCTGTAGACCTTTATCGTCGT2638,4659,9
monkeypox-2500_5_RIGHT1ACTGCTAGAATCCGGTTCAGATG2347,8360,43
monkeypox-2500_7_LEFT1CACTGTAAGCATGTCCGTACCA225060,53
monkeypox-2500_7_RIGHT1TGAGAACGAGCTCTTCAAACACT2343,4860,43
monkeypox-2500_9_LEFT1AGGCTATGTTTCGCCCATCATC225060,99
monkeypox-2500_9_RIGHT1GTCCTTTACGATGAGCTCAAATGT2441,6759,68
monkeypox-2500_11_LEFT1TGTCACTCCATAACTACCACGC225060,27
monkeypox-2500_11_RIGHT1AGTTTCGTCGATAGTACTGTGTGT2441,6760,1
monkeypox-2500_13_LEFT1TCCTTATGAAGATGATGTTTGGCG2441,6759,74
monkeypox-2500_13_RIGHT1CCCTCCTGGAGAACGACAGTTA2254,5561,33
monkeypox-2500_15_LEFT1TGGAAGCGAATGATCCGGAAAA2245,4560,8
monkeypox-2500_15_RIGHT1TCCGTGGTTTCTAGTGGGTGTA225060,94
monkeypox-2500_17_LEFT1ACCTTGGCTGTCTCATTCAATAGG2445,8360,95
monkeypox-2500_17_RIGHT1TGAATGGCTGTCGTCAAAAGGT2245,4560,93
monkeypox-2500_19_LEFT1AGGCTTCCAAAAATTTTTCATCCGT253660,84
monkeypox-2500_19_RIGHT1ACGTCGCTGTAATAGACAAGGC225060,91
monkeypox-2500_21_LEFT1CCCTAGGACGAACTACTGCCAT2254,5561,46
monkeypox-2500_21_RIGHT1TTGTGCTGCTCTTATCGTCTGA2245,4559,95
monkeypox-2500_23_LEFT1AAAAACCCTAGTATTCTTCCATCGC254059,79
monkeypox-2500_23_RIGHT1AACGGTATGTTACGGTTTGCCA2245,4560,99
monkeypox-2500_25_LEFT1CTCGCCATTTCGACATCTGGAT225060,98
monkeypox-2500_25_RIGHT1CGGGACCAAATGTAGTCAAGCT225060,8
monkeypox-2500_27_LEFT1ACGCGTTCACTATCTCCAGAGA225061,12
monkeypox-2500_27_RIGHT1TACGCACGCTTCTCCTACCTTA225061,12
monkeypox-2500_29_LEFT1TTGACTTTTTGGTCCACTTTTCCA2437,559,8
monkeypox-2500_29_RIGHT1ATATTCGTGACACTGTGCAACG2245,4559,76
monkeypox-2500_31_LEFT1TCCGGACATGATGGTAAAGACC225060,01
monkeypox-2500_31_RIGHT1AACGAATTCTGCGTCTCGTTCA2245,4561,04
monkeypox-2500_33_LEFT1TAGGCTCACCGATGATCATTGG225060,14
monkeypox-2500_33_RIGHT1AACACAGCATCCAACTGAGCAT2245,4561
monkeypox-2500_35_LEFT1ACAGGGGCAATGTTTACCACAA2245,4560,88
monkeypox-2500_35_RIGHT1CTAGACGCCACGGGGTTTAAAA225061,05
monkeypox-2500_37_LEFT1TTGTTTCGTCAACAAGTTGGATGA2437,559,92
monkeypox-2500_37_RIGHT1CGGATACCAGAGTGATAATTTCGGT254460,89
monkeypox-2500_39_LEFT1CCGCATTGGTGTTCCGATCTTA225061,18
monkeypox-2500_39_RIGHT1TGAACCTGAGGCATGGAAAAGG225061
monkeypox-2500_41_LEFT1CCACAGATTCCAATTATCAGTTGGC254460,83
monkeypox-2500_41_RIGHT1AGACGACTCTCCAAAGATAATTGGT254060,2
monkeypox-2500_43_LEFT1TGTACAGGTACCTCCATCATTAGGA254460,73
monkeypox-2500_43_RIGHT1TTGGTTGTCGACTTCCCAGTTG225061,18
monkeypox-2500_45_LEFT1TCCTGAAAACGATGATGGCAATC2343,4859,87
monkeypox-2500_45_RIGHT1AACTCTTCGAAGTGAGGATCGAT2343,4859,56
monkeypox-2500_47_LEFT1CTCCCGGATCACGATTTTGTCT225060,6
monkeypox-2500_47_RIGHT1GAACATATAGCGACGCCACCAA225061,23
monkeypox-2500_49_LEFT1TTGCATCTACATCATCCGTGGA2245,4559,75
monkeypox-2500_49_RIGHT1AATGGAAGCCGTGGTCAATAGC225061,45
monkeypox-2500_51_LEFT1TCTCTGTAGTCGACGCTCTCAA225060,79
monkeypox-2500_51_RIGHT1ACGGCCGGAAATAGTTAAGAGAC2347,8360,68
monkeypox-2500_53_LEFT1GTTGTATGGCATTGCGCAGAAA2245,4560,85
monkeypox-2500_53_RIGHT1CAAGGATGGTGTTTGTGTTGGC225060,98
monkeypox-2500_55_LEFT1CTGACAATGTACTGGGCCATGT225060,8
monkeypox-2500_55_RIGHT1ACATCATCGGAGGATAATACGCTAA254059,96
monkeypox-2500_57_LEFT1TTGGGAGAACTTAAGCGGCAAG225061,31
monkeypox-2500_57_RIGHT1AAACGATAAGAGTGGCCGCTTG225061,68
monkeypox-2500_59_LEFT1AAGATTGCGGCTAATTGCTTCG2245,4560,4
monkeypox-2500_59_RIGHT1GAGGGAATTGACTCGCGAAAGA225060,85
monkeypox-2500_61_LEFT1ACAGAACAATTAGAGCGGCAGG225061,12
monkeypox-2500_61_RIGHT1ACACGATGCGACAATGTATAGACT2441,6760,52
monkeypox-2500_63_LEFT1GACGATGATGATTGATCACTATTACACA2835,7160,19
monkeypox-2500_63_RIGHT1AATCCATCCATTGCCGTCTGAT2245,4560,34
monkeypox-2500_65_LEFT1TCAATCCCAAACCCAAAACCGT2245,4561,14
monkeypox-2500_65_RIGHT1CCCAGTAAGCAACTCCATAGCA225060,28
monkeypox-2500_67_LEFT1ACTTTCGAGGTTATTGGTTGTGGA2441,6760,77
monkeypox-2500_67_RIGHT1GCATACGCTACTCCAGAGAACG2254,5561,03
monkeypox-2500_69_LEFT1TGATGCACTAACGAGAAAATTAGAAGG2737,0460,42
monkeypox-2500_69_RIGHT1ACTTAAACCACCATCAAAAATCCATGT2733,3360,7
monkeypox-2500_71_LEFT1GGTGGAGTCGTTAAAGGTGACA225060,4
monkeypox-2500_71_RIGHT1TGCCTTGCATGTGATAAGACCT2245,4560,21
monkeypox-2500_73_LEFT1ATTGGATTCACGGTGGGTCATG225061,13
monkeypox-2500_73_RIGHT1TCACAGACAGCATTTGGATCCA2245,4560,14
monkeypox-2500_75_LEFT1ATTCGATCGTCATGGGCATAGT2245,4559,88
monkeypox-2500_75_RIGHT1TGTATCTGAATCCATGTTAGTAGTAAGCA2934,4860,78
monkeypox-2500_77_LEFT1GTTGGGACTGACAGATGTGTTCT2347,8360,75
monkeypox-2500_77_RIGHT1TGTATCGCATTCCACCCTTTCC225060,86
monkeypox-2500_79_LEFT1GATAGATCAGTGGGTGTCCATGAT2445,8360,04
monkeypox-2500_79_RIGHT1GTGTTGGGTACGACCGCTTATA225060,34
monkeypox-2500_81_LEFT1CACCTGATGGTCTGGACATACC2254,5560,34
monkeypox-2500_81_RIGHT1ACTACGTCCTTTTGCCATTGCA2245,4561,26
monkeypox-2500_83_LEFT1CCACATTGGCTAGAGGAATGCC2254,5561,51
monkeypox-2500_83_RIGHT1TGATAAGCGACGCCATTCATGT2245,4560,92
monkeypox-2500_85_LEFT1ACTAAATCTCCTTCATGCTCTCTCAC2642,3160,85
monkeypox-2500_85_RIGHT1ACCTGCTCGGTTACTTCTGTGT225061,79
monkeypox-2500_87_LEFT1CCAAGCTAAGCGACTACCATCT225060,08
monkeypox-2500_87_RIGHT1TGATGCAATTGTCTGACAACCTAGA254060,9
Primers for Pool 1

namepoolseqsize%gctm
monkeypox-2500_2_LEFT2TGTTCTACACCCTGATGCTCCT225061,01
monkeypox-2500_2_RIGHT2TCCACCCACCTTTCTTGAAATGA2343,4860,38
monkeypox-2500_4_LEFT2GTAGCAGTAGTTGGTGCATGGT225060,8
monkeypox-2500_4_RIGHT2TGTGTCCTCTCCTCTTATAACATCG254460,08
monkeypox-2500_6_LEFT2AGCGTTGACTTATGGACTCTGG225060,27
monkeypox-2500_6_RIGHT2TACCTATCCAACGACAGGCACT225061,07
monkeypox-2500_8_LEFT2TTGCGGACATGTTACACTCCTT2245,4560,41
monkeypox-2500_8_RIGHT2ACTATGGATCCCCACCACTTGA225060,75
monkeypox-2500_10_LEFT2TCGCCGTCATTTCTCCAAAGAA2245,4560,73
monkeypox-2500_10_RIGHT2TCTGTTGTTTACCACTCAGCGG225060,99
monkeypox-2500_12_LEFT2GGAACCGTTTTCGTACCGTACT225060,78
monkeypox-2500_12_RIGHT2AGTCAGGTCTTGAAGGCTACCA225060,95
monkeypox-2500_14_LEFT2TGATCCAAACCCTTGATCTCCTC2347,8360,06
monkeypox-2500_14_RIGHT2ACGGATTTCAGATGGCCATTGA2245,4560,54
monkeypox-2500_16_LEFT2GGCTGCTCCTGTTCTTGTAGTC2254,5561,11
monkeypox-2500_16_RIGHT2GATAACGCCAAAATCGCTGCTC225061,03
monkeypox-2500_18_LEFT2AAATTCGCGCCCACAATTCATC2245,4560,91
monkeypox-2500_18_RIGHT2TCGCCGTTTCATTTTCAACAGC2245,4561,03
monkeypox-2500_20_LEFT2AGAAATGCCAAATCTATAAGAAAAGTCCT2931,0360,27
monkeypox-2500_20_RIGHT2CCTTTATCAACAAGGAAAGCGTGT2441,6760,34
monkeypox-2500_22_LEFT2TCGTATTGTGGTTATATGGCTACAATT2733,3359,56
monkeypox-2500_22_RIGHT2TGAATTGTTGCAACGGTTTCCA2240,9160,01
monkeypox-2500_24_LEFT2TCAGTCGTTCTAACTCCTTTGCT2343,4859,93
monkeypox-2500_24_RIGHT2CACGCTTCTATGTTGCCGTCTA225060,91
monkeypox-2500_26_LEFT2AGACAGAATATCGTGAACAGGTGG2445,8360,7
monkeypox-2500_26_RIGHT2TGTTTCGACTGGAGAATCATCCA2343,4859,99
monkeypox-2500_28_LEFT2TAACTCCAGGCCGTTTGTTTCC225061,25
monkeypox-2500_28_RIGHT2TTGTGTACCAGAACTCCACCTAAA2441,6759,92
monkeypox-2500_30_LEFT2CTGCCACGTTAGAGGATGACAG2254,5560,92
monkeypox-2500_30_RIGHT2ACTAACGTTTCTTAGCGGAGGC225060,85
monkeypox-2500_32_LEFT2CAAGACGTTAGAGACAAGAGACGT2445,8360,63
monkeypox-2500_32_RIGHT2CAACGCCACAGATTTCTGGAGA225061,05
monkeypox-2500_34_LEFT2GCTATTTAAATGGGTGCCGCAG225060,72
monkeypox-2500_34_RIGHT2GGTGATGATCCTTGACGGAAGA225060,01
monkeypox-2500_36_LEFT2GGCCGCCATCATGATCCTATTC2254,5561,44
monkeypox-2500_36_RIGHT2TTACCGCCTTCTGGATAACCTG225060,01
monkeypox-2500_38_LEFT2AGGTGGTGGAACTCCTATTGGA225060,68
monkeypox-2500_38_RIGHT2CACCGCTTCGAAACCATGAAAC225061,09
monkeypox-2500_40_LEFT2TCACGTCAGCGGCATCTAAATT2245,4560,86
monkeypox-2500_40_RIGHT2TTCATGTGAAACTTTGTCCTTTCCT253659,73
monkeypox-2500_42_LEFT2AGCCCGTAAATGCAATCAGTGA2245,4560,8
monkeypox-2500_42_RIGHT2GCCGTTAAACCAAGCGAATACA2245,4560,02
monkeypox-2500_44_LEFT2ACGTGTACTGTATCGACCGGAT225061,18
monkeypox-2500_44_RIGHT2ACGGGTTCAGAAATATCGACGT2245,4560,01
monkeypox-2500_46_LEFT2CCAAGATCAAAAGACACGCACG225060,84
monkeypox-2500_46_RIGHT2TTGATGATGTGGAAGGGTCTGC225060,8
monkeypox-2500_48_LEFT2AGATGGGCCCGTTCTCTGAATA225061,15
monkeypox-2500_48_RIGHT2TGTAGCTGTTGTAGACATAACGGTA254059,97
monkeypox-2500_50_LEFT2GCTACTTCGTCGATGGAAACCA225060,85
monkeypox-2500_50_RIGHT2TCCTTAAATCTGGTGCCGTTGT2245,4560,41
monkeypox-2500_52_LEFT2AACCAAAAAGTCACACGCTCCA2245,4561,38
monkeypox-2500_52_RIGHT2TTCTATGCAGGATCTCCCGAAG225059,55
monkeypox-2500_54_LEFT2GAGAACATAATGCCGCCGTAGT225060,98
monkeypox-2500_54_RIGHT2TGACGTACATCCAGGAGAACCT225060,74
monkeypox-2500_56_LEFT2CACACACGGCAGAAAAACCATC225061,03
monkeypox-2500_56_RIGHT2GTTCCGTTCCCATCATAGTCGT225060,34
monkeypox-2500_58_LEFT2GAAACGGAATCGGTAGATCGTCT2347,8360,49
monkeypox-2500_58_RIGHT2CATAGCGTCTCCGGATTCCAAG2254,5561,04
monkeypox-2500_60_LEFT2ACTCGACGAGCTCACGTTTAAG225060,84
monkeypox-2500_60_RIGHT2GTTCGACGATTAACGGAGAGCA225060,91
monkeypox-2500_62_LEFT2GCTTCGCGTTTAGTCTCTGGAT225060,91
monkeypox-2500_62_RIGHT2TCGATGCCTGTAAAGGGGAAAC225060,8
monkeypox-2500_64_LEFT2ACCATCATCATAGCATGCGACT2245,4560,14
monkeypox-2500_64_RIGHT2GTGTTTGGTTGCGTTATTGCCA2245,4560,98
monkeypox-2500_66_LEFT2TAATAAGTTCGAGGATGCCGCC225060,73
monkeypox-2500_66_RIGHT2TTTTCCATGGACTTGTTCAACGT2339,1359,56
monkeypox-2500_68_LEFT2ATGTCTCGTGGGGCATTAATCG225060,99
monkeypox-2500_68_RIGHT2ACCGGATTCATCGTCGTAACAA2245,4560,27
monkeypox-2500_70_LEFT2AGACTAGTGTATGTGGAAATGTCATAGA2835,7160,24
monkeypox-2500_70_RIGHT2TCGGATTATAGCTAAGGACTAGATTCG2740,7460,21
monkeypox-2500_72_LEFT2GCAAAAATCAATGGGTCGTTGGAC2445,8361,93
monkeypox-2500_72_RIGHT2GTGACACCCATTCATCTGGAGA225059,95
monkeypox-2500_74_LEFT2TCCTTTTAGTGCTCGACAGTGT2245,4559,82
monkeypox-2500_74_RIGHT2ACATTGTTTGCCACGTCTTGAT2240,9159,56
monkeypox-2500_76_LEFT2TCTTCCGATATCTACAAGGATATTCCA2737,0459,61
monkeypox-2500_76_RIGHT2ACGGATGATCTGCACAGAACTC225060,6
monkeypox-2500_78_LEFT2CTCATGTTCTTGTGTAATCGCAGT2441,6759,92
monkeypox-2500_78_RIGHT2TGTTCTGCGTCATCTACATCTGA2343,4859,81
monkeypox-2500_80_LEFT2AGCGAGAGATCTAGCAACTAGAGT2445,8360,77
monkeypox-2500_80_RIGHT2TCGAGTCATTTTACGCACGGTT2245,4561,04
monkeypox-2500_82_LEFT2GCTCAATCTGCCAGGATCAAGT225060,87
monkeypox-2500_82_RIGHT2TCAATGGAGCAGGAAAATGGGT2245,4560,41
monkeypox-2500_84_LEFT2GACCTCACAACACAGTGCAAGA225061,18
monkeypox-2500_84_RIGHT2CCAGCTAACATAAGAGCCAATCTCA254461,07
monkeypox-2500_86_LEFT2AAAACCATGATGTGATAAAGCTCTGT2634,6260,01
monkeypox-2500_86_RIGHT2CCATTGGATGGTGCATGTGGT2152,3861,34
monkeypox-2500_88_LEFT2CCGGGAACTTACGCTTTCAGAT225060,86
monkeypox-2500_88_RIGHT2AAAAATGTGTGACCCACGACCG225062,07
Primers pool 2


If you have ordered each primer independently and need to generate primer pool stocks: add Amount5 µL of each primer from Pool 1 to a Amount1.5 mL Eppendorf labeled “Pool 1 (100µM)” and each primer from Pool 2 to a Amount1.5 mL Eppendorf labelled “Pool 2 (100µM)”. These are your 100µM stocks of each primer pool.

Note
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.

Multiplex PCR
Multiplex PCR
42m
42m
In the mastermix hood set up the multiplex PCR mastermix as follows in an Amount1.5 mL Eppendorf PCR tube:

Components volume (pool 1) Volume (pool 2)
Nuclease Free Water Amount3.94 µL Amount3.94 µL
Primer Pool 1 (100µM) Amount1.06 µL
Primer Pool 2 (100µM) Amount1.06 µL
NextGenPCR™ Arctic Fox HF Chemistry-2x Amount10 µL Amount10 µL
Total Amount15 µL Amount15 µL

Add Amount5 µL of each DNA sample to the NextGenPCR microplate containing Amount15 µL Pool 1. Mix well by pipetting
Add Amount5 µL of each DNA sample to the NextGenPCR microplate containing Amount15 µL Pool 2. Mix well by pipetting

Note
The extraction and sample addition cabinet should be cleaned with decontamination wipes and UV sterilized before and after use.


Set-up the following program on the NextGenPCR™ Instrument #10001, (Molecular Biology Systems B.V., The Netherlands):
Step Temperature Time Cycles
Heat Activation Temperature98 °C Duration00:01:00 1
Denaturation Temperature98 °C Duration00:00:10 35
Annealing and Extension Temperature65 °C Duration00:01:00 35
Total PCR time is 42 minutes

42m
Pooling
Pooling
Label a Amount0.2 mL PCR tube for each sample and combine the two pools from the individual PCR reaction as follows:

Component Volume
Pool 1 PCR reaction Amount10 µL
Pool 2 PCR reaction Amount10 µL
Total Amount20 µL

individual sample Bead cleaning (Optional)
individual sample Bead cleaning (Optional)
Ampure XP Bead Cleanup. Add a total of Amount30 µL of beads to Amount20 µL of pooled samples.

Vortex or resuspend beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
Incubate for 5 minutes at room temperature
Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear.
2m
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add Amount150 µL of freshly prepared room-temperature Concentration80 % volume volume ethanol to the pellet.

Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and re-form a pellet. Remove the ethanol using a pipette and discard.

Repeat step 7.5 and 7.6
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette
With the tube lid open incubate for Duration00:01:00 or until the pellet loses it's shine ( if the pellet dries completely it will crack and become difficult to resuspend)

1m
Remove the tube from the magnetic rack. Resuspend Pellet in Amount20 µL Nuclease free water mix gently by flicking and incubate at room temperature for Duration00:02:00

2m
Place on magnet for beads to collect on the magnetic side of the holder and the supernatant to become clear and then transfer Amount18 µL sample to a clean 96 wells Microplate ensuring no beads are transferred into this tube.

Quantification
Quantification
Prepare a mastermix of Qubit™ working solution for the required number of samples and standards. The Qubit dsDNA kit requires 2 standards for calibration.


Per sample:
Qubit® dsDNA HS Reagent Amount1 µL µL
Qubit® dsDNA HS Buffer Amount199 µL µL

Aliquot Qubit™ working solution to each tube:
  • standard tubes requires 190µL of Qubit™ working solution
  • sample tubes require anywhere from Amount180 µL t to Amount199 µL (depending how much sample you wish to add)
The final volume in each tube must be 200µL once sample/standard has been added.


Add 10µL of standard to the appropriate tube.


Add 1–20µL of each user sample to the appropriate tube.
Mix each tube vigorously by vortexing for 3–5 seconds.

Allow all tubes to incubate at room temperature for 2 minutes, then proceed to “Read standards and samples”.
On the Home screen of the Qubit™ 3 Fluorometer, press DNA, then select 1X dsDNA HS as the assay type. The Read standards screen is displayed. Press Read Standards to proceed.

Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read standard. When the reading is complete (~3 seconds), remove Standard #1.

Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read standard. When the reading is complete, remove Standard #2.

The instrument displays the results on the Read standard screen. For information on interpreting the calibration results, refer to the Qubit™ Fluorometer User Guide, available for download at thermofisher.com/qubit.

Press Run samples.

On the assay screen, select the sample volume and units:
  • Press the + or – buttons on the wheel, or anywhere on the wheel itself, to select the sample volume added to the assay tube (from 1–20µL).
  • From the unit dropdown menu, select the units for the output sample concentration (in this case choose ng/µL).
Insert a sample tube into the sample chamber, close the lid, then press Read tube. When the reading is complete (~3 seconds), remove the sample tube.
The top value (in large font) is the concentration of the original sample and the bottom value is the dilution concentration. For information on interpreting the sample results, refer to the Qubit™ Fluorometer User Guide.
Carefully record all results and store run file from the Qubit on a memory stick.
All negative controls should ideally be ‘too low’ to read on the Qubit machine, but MUST be < 1ng per ul. If your negative controls >1ng per ul, considerable contamination has occurred and you must redo previous steps.
Normalisation
Normalisation
Adjust the amount of DNA in the tube to be 100 ng total per sample in 7.5 µL molecular grade water. For example if your PCR reaction is at 100ng/ul, add 1ul of the PCR reaction to 6.5ul of molecular grade water. Input to the Rapid Barcoding kit will vary depending on the amplicon length but we have determined 50-200 ng works for efficient barcoding of this amplicon length. If there is under 100ng or you do not know the concentration, simply use all 7.5 µL of the pooled PCR reaction. Use the full 7.5 µLof the negative control, even if there is no detectable DNA in the PCR reaction.
Rapid barcoding
Rapid barcoding
9m 30s
9m 30s
Add Amount7.5 µL of each diluted PCR reaction to the labeled PCR tube.
Set up the following reaction from each sample:

Component Volume
DNA amplicons Amount7.5 µL
Fragmentation Mix RB01-12 Amount2.5 µL
Total Amount10 µL

Mix gently by flicking the tube, and spin down.
Incubate the reaction in a PCR machine:
Temperature30 °C for Duration00:01:00
Temperature80 °C for Duration00:01:00
Temperature4 °C for Duration00:00:30

2m 30s
Pool all barcoded samples, noting the total volume
Bead Cleanup. Use a 1:1 ratio of sample to beads.

Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add Amount50 µL room temperature SPRI beads to a Amount50 µL reaction.

Pulse centrifuge to collect all liquid at the bottom of the tube.

Incubate for Duration00:05:00 at room temperature.

5m
Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear.

2m
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add Amount200 µL of freshly prepared room-temperature Concentration80 % volume volume ethanol to the pellet.
Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and re-form a pellet. Remove the ethanol using a pipette and discard.

And repeat ethanol wash (steps 11.7-11.8)
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette
With the tube lid open incubate for Duration00:01:00 or until the pellet loses it's shine ( if the pellet dries completely it will crack and become difficult to resuspend)
1m
Remove the tube from the magnetic rack. Resuspend Pellet in Amount10 µL 10 mM Tris-HCl pH 8.0 with 50 mM NaCl, mix gently by flicking and incubate at room temperature for Duration00:02:00

2m
Place on magnet and transfer sample to a clean Amount1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.

Add Amount1 µL of RAP (from the SQK-RBK110.96 kit) to Amount10 µL cleaned, barcoded DNA, mix gently by flicking the tube, and spin down.
Incubate the reaction for Duration00:05:00 at room temperature.
5m
The prepared library is used for loading into the MinION flow cell according to Oxford Nanopore Rapid Barcoding (SQK-RBK110.96) protocol. Please refer to the Oxford Nanopore Rapid Barcoding SQK-RBK110.96 protocol at this stage. Store the library on ice until ready to load.
MinION sequencing
MinION sequencing
Start the sequencing run using MinKNOW.