TITLE: Monkeypox virus whole genome sequencing using combination of NextGenPCR and Oxford Nanopore
AUTHORS: Matthijs  Welkers, M.   Jonges, Anton  van den Ouden

[DESCRIPTION]
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.

[STEPS]
SECTION: Primer pool preparation
1. If required, resuspend lyophilised primers to a concentration of 100µM each
 



SECTION: Multiplex PCR
4. Add 5 µL of each DNA sample to the NextGenPCR microplate containing 15 µL Pool 1.  Mix well by pipetting
Add 5 µL of each DNA sample to the NextGenPCR microplate containing 15 µL Pool 2.  Mix well by pipetting

 


SECTION: Multiplex PCR
5. Set-up the following program on the NextGenPCR™ Instrument #10001, (Molecular Biology Systems B.V., The Netherlands):



Step                                        Temperature     Time    Cycles


Heat Activation                         98 °C  1 min    1
Denaturation                             98 °C  10 s   35 
Annealing and Extension        65 °C  1 min   35

Total PCR time is 42 minutes



 
SECTION: Primer pool preparation
2. If you have ordered each primer independently and need to generate primer pool stocks: add  5 µL  of each primer from Pool 1 to a 1.5 mL Eppendorf labeled “Pool 1 (100µM)” and each primer from Pool 2 to a 1.5 mL  Eppendorf labelled  “Pool 2 (100µM)”. These are your 100µM stocks of each primer pool.

 

SECTION: Primer pool preparation
1.1. Primers used to generate 2500 bp amplicons are here:
 
                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    monkeypox-2500_40_LEFT  2  TCACGTCAGCGGCATCTAAATT  22  45,45  60,86    monkeypox-2500_40_RIGHT  2  TTCATGTGAAACTTTGTCCTTTCCT  25  36  59,73    monkeypox-2500_42_LEFT  2  AGCCCGTAAATGCAATCAGTGA  22  45,45  60,8    monkeypox-2500_42_RIGHT  2  GCCGTTAAACCAAGCGAATACA  22  45,45  60,02    monkeypox-2500_44_LEFT  2  ACGTGTACTGTATCGACCGGAT  22  50  61,18    monkeypox-2500_44_RIGHT  2  ACGGGTTCAGAAATATCGACGT  22  45,45  60,01    monkeypox-2500_46_LEFT  2  CCAAGATCAAAAGACACGCACG  22  50  60,84    monkeypox-2500_46_RIGHT  2  TTGATGATGTGGAAGGGTCTGC  22  50  60,8    monkeypox-2500_48_LEFT  2  AGATGGGCCCGTTCTCTGAATA  22  50  61,15    monkeypox-2500_48_RIGHT  2  TGTAGCTGTTGTAGACATAACGGTA  25  40  59,97    monkeypox-2500_50_LEFT  2  GCTACTTCGTCGATGGAAACCA  22  50  60,85    monkeypox-2500_50_RIGHT  2  TCCTTAAATCTGGTGCCGTTGT  22  45,45  60,41    monkeypox-2500_52_LEFT  2  AACCAAAAAGTCACACGCTCCA  22  45,45  61,38    monkeypox-2500_52_RIGHT  2  TTCTATGCAGGATCTCCCGAAG  22  50  59,55    monkeypox-2500_54_LEFT  2  GAGAACATAATGCCGCCGTAGT  22  50  60,98    monkeypox-2500_54_RIGHT  2  TGACGTACATCCAGGAGAACCT  22  50  60,74    monkeypox-2500_56_LEFT  2  CACACACGGCAGAAAAACCATC  22  50  61,03    monkeypox-2500_56_RIGHT  2  GTTCCGTTCCCATCATAGTCGT  22  50  60,34    monkeypox-2500_58_LEFT  2  GAAACGGAATCGGTAGATCGTCT  23  47,83  60,49    monkeypox-2500_58_RIGHT  2  CATAGCGTCTCCGGATTCCAAG  22  54,55  61,04    monkeypox-2500_60_LEFT  2  ACTCGACGAGCTCACGTTTAAG  22  50  60,84    monkeypox-2500_60_RIGHT  2  GTTCGACGATTAACGGAGAGCA  22  50  60,91    monkeypox-2500_62_LEFT  2  GCTTCGCGTTTAGTCTCTGGAT  22  50  60,91    monkeypox-2500_62_RIGHT  2  TCGATGCCTGTAAAGGGGAAAC  22  50  60,8    monkeypox-2500_64_LEFT  2  ACCATCATCATAGCATGCGACT  22  45,45  60,14    monkeypox-2500_64_RIGHT  2  GTGTTTGGTTGCGTTATTGCCA  22  45,45  60,98    monkeypox-2500_66_LEFT  2  TAATAAGTTCGAGGATGCCGCC  22  50  60,73    monkeypox-2500_66_RIGHT  2  TTTTCCATGGACTTGTTCAACGT  23  39,13  59,56    monkeypox-2500_68_LEFT  2  ATGTCTCGTGGGGCATTAATCG  22  50  60,99    monkeypox-2500_68_RIGHT  2  ACCGGATTCATCGTCGTAACAA  22  45,45  60,27    monkeypox-2500_70_LEFT  2  AGACTAGTGTATGTGGAAATGTCATAGA  28  35,71  60,24    monkeypox-2500_70_RIGHT  2  TCGGATTATAGCTAAGGACTAGATTCG  27  40,74  60,21    monkeypox-2500_72_LEFT  2  GCAAAAATCAATGGGTCGTTGGAC  24  45,83  61,93    monkeypox-2500_72_RIGHT  2  GTGACACCCATTCATCTGGAGA  22  50  59,95    monkeypox-2500_74_LEFT  2  TCCTTTTAGTGCTCGACAGTGT  22  45,45  59,82    monkeypox-2500_74_RIGHT  2  ACATTGTTTGCCACGTCTTGAT  22  40,91  59,56    monkeypox-2500_76_LEFT  2  TCTTCCGATATCTACAAGGATATTCCA  27  37,04  59,61    monkeypox-2500_76_RIGHT  2  ACGGATGATCTGCACAGAACTC  22  50  60,6    monkeypox-2500_78_LEFT  2  CTCATGTTCTTGTGTAATCGCAGT  24  41,67  59,92    monkeypox-2500_78_RIGHT  2  TGTTCTGCGTCATCTACATCTGA  23  43,48  59,81    monkeypox-2500_80_LEFT  2  AGCGAGAGATCTAGCAACTAGAGT  24  45,83  60,77    monkeypox-2500_80_RIGHT  2  TCGAGTCATTTTACGCACGGTT  22  45,45  61,04    monkeypox-2500_82_LEFT  2  GCTCAATCTGCCAGGATCAAGT  22  50  60,87    monkeypox-2500_82_RIGHT  2  TCAATGGAGCAGGAAAATGGGT  22  45,45  60,41    monkeypox-2500_84_LEFT  2  GACCTCACAACACAGTGCAAGA  22  50  61,18    monkeypox-2500_84_RIGHT  2  CCAGCTAACATAAGAGCCAATCTCA  25  44  61,07    monkeypox-2500_86_LEFT  2  AAAACCATGATGTGATAAAGCTCTGT  26  34,62  60,01    monkeypox-2500_86_RIGHT  2  CCATTGGATGGTGCATGTGGT  21  52,38  61,34    monkeypox-2500_88_LEFT  2  CCGGGAACTTACGCTTTCAGAT  22  50  60,86    monkeypox-2500_88_RIGHT  2  AAAAATGTGTGACCCACGACCG  22  50  62,07  
 


SECTION: Pooling
6. Label a 0.2 mL PCR tube for each sample and combine the two pools from the individual PCR reaction as follows:

Component                     Volume
Pool 1 PCR reaction        10 µL    
Pool 2 PCR reaction        10 µL 
Total                                  20 µL 

SECTION: individual sample Bead cleaning (Optional)
7. Ampure XP Bead Cleanup. Add a total of 30 µL  of beads to 20 µL  of pooled samples.

SECTION: Normalisation
9. 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. 
SECTION: Rapid barcoding
10. Add 7.5 µL of each diluted PCR reaction to the labeled PCR tube. 
Set up the following reaction from each sample:

Component                                            Volume
DNA amplicons                                  7.5 µL 
Fragmentation Mix RB01-12            2.5 µL 
Total                                                     10 µL 

SECTION: Rapid barcoding
11. Bead Cleanup. Use a 1:1 ratio of sample to beads.

SECTION: Rapid barcoding
10.1. Mix gently by flicking the tube, and spin down.
SECTION: Rapid barcoding
10.2. Incubate the reaction in a PCR machine:
30 °C for 1 min 
80 °C for 1 min 
4 °C   for 30 s 

SECTION: Rapid barcoding
10.3. Pool all barcoded samples, noting the total volume
SECTION: Rapid barcoding
11.1. Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
SECTION: Rapid barcoding
11.2. Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add 50 µL  room temperature SPRI beads to a  50 µL reaction.

SECTION: Rapid barcoding
11.3. Pulse centrifuge to collect all liquid at the bottom of the tube.

SECTION: Rapid barcoding
11.4. Incubate for 5 min   at room temperature.

SECTION: Rapid barcoding
11.5. Place on magnetic rack and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear.

SECTION: Rapid barcoding
11.6. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: MinION sequencing
12. Start the sequencing run using MinKNOW.

SECTION: Multiplex PCR
3. In the mastermix hood set up the multiplex PCR mastermix as follows in an 1.5 mL  Eppendorf PCR tube:

Components                                                            volume (pool 1)            Volume (pool 2)
Nuclease Free Water                                                3.94 µL                 3.94 µL 
Primer Pool 1  (100µM)                                           1.06 µL 
Primer Pool 2  (100µM)                                                                                    1.06 µL 
NextGenPCR™ Arctic Fox HF Chemistry-2x          10 µL                     10 µL 
Total                                                                            15 µL                     15 µL 

SECTION: individual sample Bead cleaning (Optional)
7.1. Vortex or resuspend beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.
SECTION: individual sample Bead cleaning (Optional)
7.2. Incubate for 5 minutes at room temperature
SECTION: individual sample Bead cleaning (Optional)
7.3. Place on magnetic rack and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear.
SECTION: individual sample Bead cleaning (Optional)
7.4. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: individual sample Bead cleaning (Optional)
7.5. Add 150 µL  of freshly prepared room-temperature 80 % volume  volume ethanol to the pellet.

SECTION: individual sample Bead cleaning (Optional)
7.6. 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.  

SECTION: individual sample Bead cleaning (Optional)
7.7. Repeat step 7.5 and 7.6
SECTION: individual sample Bead cleaning (Optional)
7.8. 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
SECTION: individual sample Bead cleaning (Optional)
7.9. With the tube lid open incubate for 1 min or until the pellet loses it's shine ( if the pellet dries completely it will crack and become difficult to resuspend)

SECTION: individual sample Bead cleaning (Optional)
7.10. Remove the tube from the magnetic rack. Resuspend Pellet in 20 µL Nuclease free water mix gently by flicking and incubate at room temperature for 2 min 

SECTION: individual sample Bead cleaning (Optional)
7.11. Place on magnet for beads to collect on the magnetic side of the holder and the supernatant to become clear and then transfer 18 µL sample to a clean 96 wells Microplate ensuring no beads are transferred into this tube.

SECTION: Rapid barcoding
11.7. Add 200 µL  of freshly prepared room-temperature 80 % volume  volume ethanol to the pellet.
SECTION: Rapid barcoding
11.8. 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.  

SECTION: Rapid barcoding
11.9. And repeat ethanol wash (steps 11.7-11.8)
SECTION: Rapid barcoding
11.10. 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
SECTION: Rapid barcoding
11.11. With the tube lid open incubate for 1 min or until the pellet loses it's shine ( if the pellet dries completely it will crack and become difficult to resuspend)
SECTION: Rapid barcoding
11.12. Remove the tube from the magnetic rack. Resuspend Pellet in 10 µL 10 mM Tris-HCl pH 8.0 with 50 mM NaCl, mix gently by flicking and incubate at room temperature for 2 min 

SECTION: Rapid barcoding
11.13. Place on magnet and transfer sample to a clean 1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.

SECTION: Rapid barcoding
11.14. Add 1 µL  of RAP (from the SQK-RBK110.96 kit) to 10 µL cleaned, barcoded DNA, mix gently by flicking the tube, and spin down.
SECTION: Rapid barcoding
11.15. Incubate the reaction for 5 min at room temperature.
SECTION: Rapid barcoding
11.16. 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.
SECTION: Quantification
8.16. 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.
SECTION: Quantification
8.15. Carefully record all results and store run file from the Qubit on a memory stick.
SECTION: Quantification
8.14. 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.
SECTION: Quantification
8.13. 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.
SECTION: Quantification
8.12. 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).
SECTION: Quantification
8.11. Press Run samples.


SECTION: Quantification
8.10. 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.


SECTION: Quantification
8.9. 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.


SECTION: Quantification
8.8. 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.


SECTION: Quantification
8.7. 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.

SECTION: Quantification
8.6. Allow all tubes to incubate at room temperature for 2 minutes, then proceed to “Read standards and samples”.
SECTION: Quantification
8.5. Mix each tube vigorously by vortexing for 3–5 seconds.


SECTION: Quantification
8.4. Add 1–20µL of each user sample to the appropriate tube.
SECTION: Quantification
8.3. Add 10µL of standard to the appropriate tube. 



SECTION: Quantification
8.2. Aliquot Qubit™ working solution to each tube: 
standard tubes requires 190µL of Qubit™ working solution
sample tubes require anywhere from  180 µL to 199 µL(depending how much sample you wish to add)
The final volume in each tube must be 200µL once sample/standard has been added. 



SECTION: Quantification
8.1. 

Per sample: 


Qubit® dsDNA HS Reagent 1 µL µL
Qubit® dsDNA HS Buffer     199 µLµL

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