Monkeypox virus whole genome sequencing using combination of NextGenPCR and Oxford Nanopore

Matthijs  Welkers; M.  Jonges; antonvandenouden

Jul 12, 2022

Version 1

Monkeypox 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

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

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

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

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)

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.

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)

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   

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

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

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

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.

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

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)

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   

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.

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

Start the sequencing run using MinKNOW.


name	pool	seq	size	%gc	tm
monkeypox-2500_1_LEFT	1	AAAAATGTGTGACCCACGACCG	22	50	62,07
monkeypox-2500_1_RIGHT	1	CCGGGAACTTACGCTTTCAGAT	22	50	60,86
monkeypox-2500_3_LEFT	1	GTTAACGATGCGCACAATCTCG	22	50	61,01
monkeypox-2500_3_RIGHT	1	TAGTGAGAGCGAGAGTGACAGT	22	50	60,47
monkeypox-2500_5_LEFT	1	AATAGTCTGTAGACCTTTATCGTCGT	26	38,46	59,9
monkeypox-2500_5_RIGHT	1	ACTGCTAGAATCCGGTTCAGATG	23	47,83	60,43
monkeypox-2500_7_LEFT	1	CACTGTAAGCATGTCCGTACCA	22	50	60,53
monkeypox-2500_7_RIGHT	1	TGAGAACGAGCTCTTCAAACACT	23	43,48	60,43
monkeypox-2500_9_LEFT	1	AGGCTATGTTTCGCCCATCATC	22	50	60,99
monkeypox-2500_9_RIGHT	1	GTCCTTTACGATGAGCTCAAATGT	24	41,67	59,68
monkeypox-2500_11_LEFT	1	TGTCACTCCATAACTACCACGC	22	50	60,27
monkeypox-2500_11_RIGHT	1	AGTTTCGTCGATAGTACTGTGTGT	24	41,67	60,1
monkeypox-2500_13_LEFT	1	TCCTTATGAAGATGATGTTTGGCG	24	41,67	59,74
monkeypox-2500_13_RIGHT	1	CCCTCCTGGAGAACGACAGTTA	22	54,55	61,33
monkeypox-2500_15_LEFT	1	TGGAAGCGAATGATCCGGAAAA	22	45,45	60,8
monkeypox-2500_15_RIGHT	1	TCCGTGGTTTCTAGTGGGTGTA	22	50	60,94
monkeypox-2500_17_LEFT	1	ACCTTGGCTGTCTCATTCAATAGG	24	45,83	60,95
monkeypox-2500_17_RIGHT	1	TGAATGGCTGTCGTCAAAAGGT	22	45,45	60,93
monkeypox-2500_19_LEFT	1	AGGCTTCCAAAAATTTTTCATCCGT	25	36	60,84
monkeypox-2500_19_RIGHT	1	ACGTCGCTGTAATAGACAAGGC	22	50	60,91
monkeypox-2500_21_LEFT	1	CCCTAGGACGAACTACTGCCAT	22	54,55	61,46
monkeypox-2500_21_RIGHT	1	TTGTGCTGCTCTTATCGTCTGA	22	45,45	59,95
monkeypox-2500_23_LEFT	1	AAAAACCCTAGTATTCTTCCATCGC	25	40	59,79
monkeypox-2500_23_RIGHT	1	AACGGTATGTTACGGTTTGCCA	22	45,45	60,99
monkeypox-2500_25_LEFT	1	CTCGCCATTTCGACATCTGGAT	22	50	60,98
monkeypox-2500_25_RIGHT	1	CGGGACCAAATGTAGTCAAGCT	22	50	60,8
monkeypox-2500_27_LEFT	1	ACGCGTTCACTATCTCCAGAGA	22	50	61,12
monkeypox-2500_27_RIGHT	1	TACGCACGCTTCTCCTACCTTA	22	50	61,12
monkeypox-2500_29_LEFT	1	TTGACTTTTTGGTCCACTTTTCCA	24	37,5	59,8
monkeypox-2500_29_RIGHT	1	ATATTCGTGACACTGTGCAACG	22	45,45	59,76
monkeypox-2500_31_LEFT	1	TCCGGACATGATGGTAAAGACC	22	50	60,01
monkeypox-2500_31_RIGHT	1	AACGAATTCTGCGTCTCGTTCA	22	45,45	61,04
monkeypox-2500_33_LEFT	1	TAGGCTCACCGATGATCATTGG	22	50	60,14
monkeypox-2500_33_RIGHT	1	AACACAGCATCCAACTGAGCAT	22	45,45	61
monkeypox-2500_35_LEFT	1	ACAGGGGCAATGTTTACCACAA	22	45,45	60,88
monkeypox-2500_35_RIGHT	1	CTAGACGCCACGGGGTTTAAAA	22	50	61,05
monkeypox-2500_37_LEFT	1	TTGTTTCGTCAACAAGTTGGATGA	24	37,5	59,92
monkeypox-2500_37_RIGHT	1	CGGATACCAGAGTGATAATTTCGGT	25	44	60,89
monkeypox-2500_39_LEFT	1	CCGCATTGGTGTTCCGATCTTA	22	50	61,18
monkeypox-2500_39_RIGHT	1	TGAACCTGAGGCATGGAAAAGG	22	50	61
monkeypox-2500_41_LEFT	1	CCACAGATTCCAATTATCAGTTGGC	25	44	60,83
monkeypox-2500_41_RIGHT	1	AGACGACTCTCCAAAGATAATTGGT	25	40	60,2
monkeypox-2500_43_LEFT	1	TGTACAGGTACCTCCATCATTAGGA	25	44	60,73
monkeypox-2500_43_RIGHT	1	TTGGTTGTCGACTTCCCAGTTG	22	50	61,18
monkeypox-2500_45_LEFT	1	TCCTGAAAACGATGATGGCAATC	23	43,48	59,87
monkeypox-2500_45_RIGHT	1	AACTCTTCGAAGTGAGGATCGAT	23	43,48	59,56
monkeypox-2500_47_LEFT	1	CTCCCGGATCACGATTTTGTCT	22	50	60,6
monkeypox-2500_47_RIGHT	1	GAACATATAGCGACGCCACCAA	22	50	61,23
monkeypox-2500_49_LEFT	1	TTGCATCTACATCATCCGTGGA	22	45,45	59,75
monkeypox-2500_49_RIGHT	1	AATGGAAGCCGTGGTCAATAGC	22	50	61,45
monkeypox-2500_51_LEFT	1	TCTCTGTAGTCGACGCTCTCAA	22	50	60,79
monkeypox-2500_51_RIGHT	1	ACGGCCGGAAATAGTTAAGAGAC	23	47,83	60,68
monkeypox-2500_53_LEFT	1	GTTGTATGGCATTGCGCAGAAA	22	45,45	60,85
monkeypox-2500_53_RIGHT	1	CAAGGATGGTGTTTGTGTTGGC	22	50	60,98
monkeypox-2500_55_LEFT	1	CTGACAATGTACTGGGCCATGT	22	50	60,8
monkeypox-2500_55_RIGHT	1	ACATCATCGGAGGATAATACGCTAA	25	40	59,96
monkeypox-2500_57_LEFT	1	TTGGGAGAACTTAAGCGGCAAG	22	50	61,31
monkeypox-2500_57_RIGHT	1	AAACGATAAGAGTGGCCGCTTG	22	50	61,68
monkeypox-2500_59_LEFT	1	AAGATTGCGGCTAATTGCTTCG	22	45,45	60,4
monkeypox-2500_59_RIGHT	1	GAGGGAATTGACTCGCGAAAGA	22	50	60,85
monkeypox-2500_61_LEFT	1	ACAGAACAATTAGAGCGGCAGG	22	50	61,12
monkeypox-2500_61_RIGHT	1	ACACGATGCGACAATGTATAGACT	24	41,67	60,52
monkeypox-2500_63_LEFT	1	GACGATGATGATTGATCACTATTACACA	28	35,71	60,19
monkeypox-2500_63_RIGHT	1	AATCCATCCATTGCCGTCTGAT	22	45,45	60,34
monkeypox-2500_65_LEFT	1	TCAATCCCAAACCCAAAACCGT	22	45,45	61,14
monkeypox-2500_65_RIGHT	1	CCCAGTAAGCAACTCCATAGCA	22	50	60,28
monkeypox-2500_67_LEFT	1	ACTTTCGAGGTTATTGGTTGTGGA	24	41,67	60,77
monkeypox-2500_67_RIGHT	1	GCATACGCTACTCCAGAGAACG	22	54,55	61,03
monkeypox-2500_69_LEFT	1	TGATGCACTAACGAGAAAATTAGAAGG	27	37,04	60,42
monkeypox-2500_69_RIGHT	1	ACTTAAACCACCATCAAAAATCCATGT	27	33,33	60,7
monkeypox-2500_71_LEFT	1	GGTGGAGTCGTTAAAGGTGACA	22	50	60,4
monkeypox-2500_71_RIGHT	1	TGCCTTGCATGTGATAAGACCT	22	45,45	60,21
monkeypox-2500_73_LEFT	1	ATTGGATTCACGGTGGGTCATG	22	50	61,13
monkeypox-2500_73_RIGHT	1	TCACAGACAGCATTTGGATCCA	22	45,45	60,14
monkeypox-2500_75_LEFT	1	ATTCGATCGTCATGGGCATAGT	22	45,45	59,88
monkeypox-2500_75_RIGHT	1	TGTATCTGAATCCATGTTAGTAGTAAGCA	29	34,48	60,78
monkeypox-2500_77_LEFT	1	GTTGGGACTGACAGATGTGTTCT	23	47,83	60,75
monkeypox-2500_77_RIGHT	1	TGTATCGCATTCCACCCTTTCC	22	50	60,86
monkeypox-2500_79_LEFT	1	GATAGATCAGTGGGTGTCCATGAT	24	45,83	60,04
monkeypox-2500_79_RIGHT	1	GTGTTGGGTACGACCGCTTATA	22	50	60,34
monkeypox-2500_81_LEFT	1	CACCTGATGGTCTGGACATACC	22	54,55	60,34
monkeypox-2500_81_RIGHT	1	ACTACGTCCTTTTGCCATTGCA	22	45,45	61,26
monkeypox-2500_83_LEFT	1	CCACATTGGCTAGAGGAATGCC	22	54,55	61,51
monkeypox-2500_83_RIGHT	1	TGATAAGCGACGCCATTCATGT	22	45,45	60,92
monkeypox-2500_85_LEFT	1	ACTAAATCTCCTTCATGCTCTCTCAC	26	42,31	60,85
monkeypox-2500_85_RIGHT	1	ACCTGCTCGGTTACTTCTGTGT	22	50	61,79
monkeypox-2500_87_LEFT	1	CCAAGCTAAGCGACTACCATCT	22	50	60,08
monkeypox-2500_87_RIGHT	1	TGATGCAATTGTCTGACAACCTAGA	25	40	60,9


name	pool	seq	size	%gc	tm
monkeypox-2500_2_LEFT	2	TGTTCTACACCCTGATGCTCCT	22	50	61,01
monkeypox-2500_2_RIGHT	2	TCCACCCACCTTTCTTGAAATGA	23	43,48	60,38
monkeypox-2500_4_LEFT	2	GTAGCAGTAGTTGGTGCATGGT	22	50	60,8
monkeypox-2500_4_RIGHT	2	TGTGTCCTCTCCTCTTATAACATCG	25	44	60,08
monkeypox-2500_6_LEFT	2	AGCGTTGACTTATGGACTCTGG	22	50	60,27
monkeypox-2500_6_RIGHT	2	TACCTATCCAACGACAGGCACT	22	50	61,07
monkeypox-2500_8_LEFT	2	TTGCGGACATGTTACACTCCTT	22	45,45	60,41
monkeypox-2500_8_RIGHT	2	ACTATGGATCCCCACCACTTGA	22	50	60,75
monkeypox-2500_10_LEFT	2	TCGCCGTCATTTCTCCAAAGAA	22	45,45	60,73
monkeypox-2500_10_RIGHT	2	TCTGTTGTTTACCACTCAGCGG	22	50	60,99
monkeypox-2500_12_LEFT	2	GGAACCGTTTTCGTACCGTACT	22	50	60,78
monkeypox-2500_12_RIGHT	2	AGTCAGGTCTTGAAGGCTACCA	22	50	60,95
monkeypox-2500_14_LEFT	2	TGATCCAAACCCTTGATCTCCTC	23	47,83	60,06
monkeypox-2500_14_RIGHT	2	ACGGATTTCAGATGGCCATTGA	22	45,45	60,54
monkeypox-2500_16_LEFT	2	GGCTGCTCCTGTTCTTGTAGTC	22	54,55	61,11
monkeypox-2500_16_RIGHT	2	GATAACGCCAAAATCGCTGCTC	22	50	61,03
monkeypox-2500_18_LEFT	2	AAATTCGCGCCCACAATTCATC	22	45,45	60,91
monkeypox-2500_18_RIGHT	2	TCGCCGTTTCATTTTCAACAGC	22	45,45	61,03
monkeypox-2500_20_LEFT	2	AGAAATGCCAAATCTATAAGAAAAGTCCT	29	31,03	60,27
monkeypox-2500_20_RIGHT	2	CCTTTATCAACAAGGAAAGCGTGT	24	41,67	60,34
monkeypox-2500_22_LEFT	2	TCGTATTGTGGTTATATGGCTACAATT	27	33,33	59,56
monkeypox-2500_22_RIGHT	2	TGAATTGTTGCAACGGTTTCCA	22	40,91	60,01
monkeypox-2500_24_LEFT	2	TCAGTCGTTCTAACTCCTTTGCT	23	43,48	59,93
monkeypox-2500_24_RIGHT	2	CACGCTTCTATGTTGCCGTCTA	22	50	60,91
monkeypox-2500_26_LEFT	2	AGACAGAATATCGTGAACAGGTGG	24	45,83	60,7
monkeypox-2500_26_RIGHT	2	TGTTTCGACTGGAGAATCATCCA	23	43,48	59,99
monkeypox-2500_28_LEFT	2	TAACTCCAGGCCGTTTGTTTCC	22	50	61,25
monkeypox-2500_28_RIGHT	2	TTGTGTACCAGAACTCCACCTAAA	24	41,67	59,92
monkeypox-2500_30_LEFT	2	CTGCCACGTTAGAGGATGACAG	22	54,55	60,92
monkeypox-2500_30_RIGHT	2	ACTAACGTTTCTTAGCGGAGGC	22	50	60,85
monkeypox-2500_32_LEFT	2	CAAGACGTTAGAGACAAGAGACGT	24	45,83	60,63
monkeypox-2500_32_RIGHT	2	CAACGCCACAGATTTCTGGAGA	22	50	61,05
monkeypox-2500_34_LEFT	2	GCTATTTAAATGGGTGCCGCAG	22	50	60,72
monkeypox-2500_34_RIGHT	2	GGTGATGATCCTTGACGGAAGA	22	50	60,01
monkeypox-2500_36_LEFT	2	GGCCGCCATCATGATCCTATTC	22	54,55	61,44
monkeypox-2500_36_RIGHT	2	TTACCGCCTTCTGGATAACCTG	22	50	60,01
monkeypox-2500_38_LEFT	2	AGGTGGTGGAACTCCTATTGGA	22	50	60,68
monkeypox-2500_38_RIGHT	2	CACCGCTTCGAAACCATGAAAC	22	50	61,09

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

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