Sep 03, 2024

Public workspaceBCCDC / ARTIC Mpox V2.3.4 2500bp amplicon generation and NGS

DOI
dx.doi.org/10.17504/protocols.io.n2bvj34nnlk5/v1
  • 1BCCDC Public Health Laboratory;
  • 2ARTIC Network;
  • 3University of Birmingham;
  • 4University of British Columbia Department of Pathology & Laboratory Medicine
BCCDC Public Health Laboratory
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj34nnlk5/v1
Protocol Citation: Anthea Lam, Chris Kent, Michael Chan, Alan O'Dwyer, Jorja M Eacrett, Tracy Lee, Frankie Tsang, Tara Newman, Dan Fornika, Natalie Prystajecky, James Zlosnik, Agatha Jassem, Josh Quick, John Tyson 2024. BCCDC / ARTIC Mpox V2.3.4 2500bp amplicon generation and NGS. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj34nnlk5/v1
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: February 02, 2024
Last Modified: September 03, 2024
Protocol Integer ID: 94639
Keywords: mpox, PCR, amplicons, artic, BCCDC
Funders Acknowledgement:
Canada-Africa Monkeypox Partnership
BCCDC Public Health Laboratory
Abstract
This procedure provides instructions on how to generate amplicons and NGS data for the near whole genome of Mpox. Illumina, Oxford Nanopore or other NGS sequencing platforms can all be utilized if amplicon specific library preparations and platform specific bioinformatic analysis pipelines are available. We detail specific use of a modified Illumina DNA prep library preparation and the analysis pipelines using the generated data in this protocol. The BCCDC / ARTIC V2.3.4 Mpox WGS 2500bp primer scheme was optimized to match and efficiently generate data from the global 2022 West African clade outbreak. The MPV_3000_1_Left to MPV_3000_79_Right primers tile the near whole genome of the Mpox virus.
Since the first and last ~6500bp of the Mpox genome (ITR) are usually identical, the last ~6500bp of the genome is masked out on the reference genome to simplify analysis using short read Illumina data. The resulting ITR regions thus represent a consensus ITR combining the amplification products from the dual ITR primer binding sites. For specific ITR region sequence information the use of unique external primer binding sites is required along with removal of all but one common internal ITR binding site to generate specific region spanning PCR products. These products can then be sequenced using Nanopore long read sequencing, see here for some candidate primer combinations (MPV_3000_4_RIGHT, MPV_3000_80_LEFT, and MPV_3000_1_LEFT), to provide specific ITR region sequences. This approach was used to generate the BCCDCmpx2 sequence (available on GISAID, EPI_ISL_13351002) which used both Oxford Nanopore and Illumina data to fully sequence the individual ITR regions and repeat regions refractory to short read technologies alone.

The sample PCRs are performed in two pools before being combined for library preparation in order to minimize primer interactions and allow near complete coverage tiling of the Mpox genome. Sequences for the primers and locations on reference genomes can be found in the Materials section tables as well as linked file locations and here.

Effort were made to increase sequencing efficiency / genome coverage for a given amount of generated sequence data by performing a number of rounds of primer pair concentration re-balancing in the PCR primer pools (Figure 1.). The primer amounts for V2.3.4 are detailed in this protocol and Materials section.


Figure 1. Iterative experimental amplicon balancing to increase sequencing efficiency for a given sample

Materials
Samples: Nucleic acid extracts previously found to be positive for Mpox virus by a validated laboratory method.


ReagentsEquipmentSupplies
Modified custom primers, see sequences below Post-PCR thermalcycler 96-well PCR plate
Q5 Hot Start High Fidelity DNA Polymerase (New England BioLabs, M0493L) Appropriate volume pipettes (single and multi-channel) Plate adhesive covers
10mM dNTPs (SuperScript IV First Strand Synthesis Kit ThermoFisher #18091200 Vortex Mixer Filtered pipet tips; various sizes
Amplirun MPXV DNA control (Somagen Diagnostics, MBC146-R) Plate centrifuge1.7mL microcentrifuge tubes
Ultrapure water Biological safety cabinet
Repeater pipet

Quality Control:
Negative control: PCR Grade Water
Positive control: Amplirun MPXV DNA control (Somagen Diagnostics, MBC146-R) [2] is purified DNA of the complete viral genome of Mpox virus.


BCCDC/ARTIC Mpox V2.3.4 Primer Scheme sequences

Mapping locations and reference specific bed files etc. for analysis pipeline can be found here.


Table 1. BCCDC/ARTIC Mpox V2.3.4 primer Pool 1 sequences and 10x stock pool amounts
ABC
Pool 1 PrimerSequenceVol 100uM (uL)
MPV_3000_1_LEFTAACTCTAGAGGGTAAGAAAAATCAATCG4
MPV_3000_1_RIGHTmodCGGGAACTTACGCTTTCAGATTATG4
MPV_3000_3_LEFTTTAACGATGCGCACAATCTCGTC2
MPV_3000_3_RIGHTAGCCACTAATAAAAACAGTTGGTTAATTACTTC2
MPV_3000_5_LEFTmodCCATGTTCGAGCATACTAGCCATG2
MPV_3000_5_RIGHTGACAGGATCGAATTCCATATCCGC2
MPV_3000_7_LEFTGATATCGCCGAATGTCATATACTCAATTAG4
MPV_3000_7_RIGHTGGATAAACTATTATCTGCCCATGATACRTTT4
MPV_3000_9_LEFTACAGTGAGTTTATCTTTCTTTGAAGTGATA8
MPV_3000_9_RIGHTCGTGATATTTTCTATCAATGGGGAAATTATTACG8
MPV_3000_11_LEFTTGCGCTGGGCTCAATGTCTTA6
MPV_3000_11_RIGHTmodAGCCATATATCTACTAATCAGATCTATTAGAGA6
MPV_3000_13_LEFTCCGCCTTTGAATAAATAATAGGAATAAAGTTCA6
MPV_3000_13_RIGHTTGAAGTTAATACTATACTAATGAACAACAAGGG6
MPV_3000_15_LEFTGTATTCACCCACACGTTTTTCGAAAAA4
MPV_3000_15_RIGHTTAATCCTCCACCAAAATAGAGTATATCATCTCA4
MPV_3000_17_LEFTCATTCAATAGGGTAGTGATATTTGTATGTATGA8
MPV_3000_17_RIGHTTCCATTGATCGATATTATACGAAAGCG8
MPV_3000_19_LEFTTAGCCCCGCAATACTCCTCAT3
MPV_3000_19_RIGHTTCAGAGTTTTAAATCCTCAAAATGAATAGG3
MPV_3000_21_LEFTGATTTTCCATCTGCCTTATCGAATACTCTT3
MPV_3000_21_RIGHTTTCCCCTCCGTGAAATAATGGATTAAG3
MPV_3000_23_LEFTATTCAGATACGTCTATACAGATAATACCAAACA4
MPV_3000_23_RIGHTTAACAGGAGAGAAGATTATCTTTAGATCTCCA4
MPV_3000_25_LEFTTCTCRACATCTTCAATAGATACCTTGCTA4
MPV_3000_25_RIGHTTCAATAGTATCAGTGCCCGTGC4
MPV_3000_27_LEFTACCACTCTCCRTCATCCTTCAC4
MPV_3000_27_RIGHTTGGACCAAAAAGTCAATTAATGTGATATGTG4
MPV_3000_29_LEFTGTTGCACAGTGTCACGAATATAAATCATATTTA6
MPV_3000_29_RIGHTGATATCTACTTGGGAATTTCTAATTTCGGATTC6
MPV_3000_31_LEFTGTTACCCTGCCAATTAATGTACGCA3
MPV_3000_31_RIGHTCTAGATGAATGACGGTTCTACCACAAC3
MPV_3000_33_LEFTTGGCGCTAGTCATCACATTAACTATTTTT3
MPV_3000_33_RIGHTTTTGAATCGCATCAAACTAATCACAAAGTC3
MPV_3000_35_LEFTmodGCTACTCGTTTGGAATCACAGACATTAT6
MPV_3000_35_RIGHTATTAAGAATATAACCTCTTCCTTCTGGATCCT6
MPV_3000_37_LEFTATCCAGTCTTTAATGAATTAACGAGATATATGC6
MPV_3000_37_RIGHTCAGCGCTTTATCTTTAATATTATCATGTCT6
MPV_3000_39_LEFTGCGGTTTTTATGTTAATGGAAACTATGTTT6
MPV_3000_39_RIGHTCTATAAACTTAATAGGTACAACAACGGACTTAA6
MPV_3000_41_LEFTTGTGTGTGGTATAGATCCGTATCCAAA6
MPV_3000_41_RIGHTTTTGTACCATTTAACCAACAAGTATAGAAATGC6
MPV_3000_43_LEFTCCGTATATAAGGCGTATCTCCATAGAG8
MPV_3000_43_RIGHTCAATAATTTGATCACTCGTTAGAGATATTCKTC8
MPV_3000_45_LEFTTTAGTGACGGACTTAACATGAGACATAAATAAA4
MPV_3000_45_RIGHTGGTTAAGTCTCACGTAGCTCTTAGAGAA4
MPV_3000_47_LEFTGCAGGAAGTTTAATACCGAATAGTGC4
MPV_3000_47_RIGHTCCTAAAATCATACTGTTTCAGAATAACGACATC4
MPV_3000_49_LEFTGTAATGTCCAAATCTAGTTGTGGAAATACTTC4
MPV_3000_49_RIGHTTCGGAACCGATAAGTATCTTGGATAGAAT4
MPV_3000_51_LEFTGTGGTTAATCTTAGAAATCAACGATCTAATACA4
MPV_3000_51_RIGHTGCATTATACGTCTTTTGTAACGACCATAAAG4
MPV_3000_53_LEFTCCAGATATGGTCAAATCRATTAAGGAACTAA12
MPV_3000_53_RIGHTTTTTCGATCTCTATATGAATTACCAGACATGA12
MPV_3000_55_LEFTGTCATCGCATCCTAACGTACGG3
MPV_3000_55_RIGHTACAGATTTCCGAATATCAGGAGCATAAC3
MPV_3000_57_LEFTGCATCATCTTTATCATTATTAGGTGGGGG6
MPV_3000_57_RIGHTGCAAATACATGCATCAGTAGAAAACATAGAAA6
MPV_3000_59_LEFTGTTGTATTGGGGAAGGTAACAATTAATGATC4
MPV_3000_59_RIGHTAYATCTAACATGATATCATAGTCGCATGC4
MPV_3000_61_LEFTCATGGCAATAACGCAACCAAACA4
MPV_3000_61_RIGHTGARCTGTATGTCAATACCGGCATTA4
MPV_3000_63_LEFTAAATGCATTAGTTTACTTTTCTACTCAGCA4
MPV_3000_63_RIGHTATTATCGTCTAATGAATTAATCAACTCGTTCTG4
MPV_3000_65_LEFTTCTACTCATCTAAACGATTTAGTAAACTTGACT6
MPV_3000_65_RIGHTAACTTAAACCACCATCAAAAATCCATGT6
MPV_3000_67_LEFTCGATAAAGAAAAAGATTGTTGGACATTGGATAA8
MPV_3000_67_RIGHTCGTCTAGATACAACGTATCCTTTATGC8
MPV_3000_69_LEFTACATAATCTCGGGTTATCATCCATTAATAT4
MPV_3000_69_RIGHTGCATCGTAGATATCAACATCCACTGAAG4
MPV_3000_71_LEFTmodTATTCCAAAATGTTCCAGAACTCTCTGTATT4
MPV_3000_71_RIGHTmodCCATTAGCTCCATAATACAGTATTGTCAATAC4
MPV_3000_73_LEFTmodGGAGACGGTAGAATAAGTGTAGCAAATAAAA4
MPV_3000_73_RIGHTCTGATCTATCATAACCATAGAATTCATCAACAA4
MPV_3000_75_LEFTATGTTACCCGATATACCCATAGCATTATCTA3.5
MPV_3000_75_RIGHTTCTCCTCGATAATAAGCCATATCTGG3.5
MPV_3000_77_LEFTCTACAAATGATGGTCTACAGAGTCCAA4
MPV_3000_77_RIGHTAATGATGAAAACAAGCACGTGGTTTATC4
MPV_3000_79_LEFTCTGGATAGACATAAATATCCTCCTCGTAATAATG12
MPV_3000_79_RIGHTGCTATCGAACCATTTTTGTAGTCTAAAGC12
Table 2. BCCDC/ARTIC Mpox V2.3.4 primer Pool 2 sequences and 10x stock pool amounts
ABC
Pool 2 PrimerSequenceVol 100uM (uL)
MPV_3000_2_LEFTCACACGGYGTACATTGTGTATTAGT4
MPV_3000_2_RIGHTTGACAATCTCCTATCAAAACTTCCATTAGAAAT4
MPV_3000_4_LEFTGATCTGAGATAAATTATACAATCTTCGCTATCGA3
MPV_3000_4_RIGHTATTCTATTGATCTAACGCTGTAYGACG3
MPV_3000_6_LEFTAAAGTTGTGGTAGTATGATCTCCATATTTA3
MPV_3000_6_RIGHTTTGAAAACAATGAACTCGGATGTCCT3
MPV_3000_8_LEFTGTACACTGTATGATAAGATACATATTCTGACAA4
MPV_3000_8_RIGHTGCTAAAGATATAAATACGATGTCATTCGAC4
MPV_3000_10_LEFTCTTTAGATGGTGATCCTGAATGTGTTTAAAAA4
MPV_3000_10_RIGHTmodAAACAAAACAAATTTGGAGATAGTCCTCTTA4
MPV_3000_12_LEFTATGTCTGAAACGAGACGCTAATTAGTG2
MPV_3000_12_RIGHTCCGCTTTATTGTCATGTATCGGAAATG2
MPV_3000_14_LEFTGGTAATGAATGAAATTGTCGCCAATCT3
MPV_3000_14_RIGHTAATGTTACCAATCCGTTTGATTTCATGG3
MPV_3000_16_LEFTAGTAAAAAGTTCACATTGAAACTATGTCAGTAG6
MPV_3000_16_RIGHTTTCTGCATTATTGTAATTCGTATGCTGATAC6
MPV_3000_18_LEFTCCGTATCGTTCTAAAGCCAGTGC4
MPV_3000_18_RIGHTGATGAACAAAATGGGYATTCTAGACAAAAA4
MPV_3000_20_LEFTCTCTGCCTAAATAGGTTTCTATAATCTTTAATG2
MPV_3000_20_RIGHTAACTCTCCTACATTTTATGCCTGTGTAG2
MPV_3000_22_LEFTGACGAATTAGGAGAACTTATAGGCGTAAATTA3
MPV_3000_22_RIGHTTTATAGTCTCCTGTTTGATGTATATACCTAGC3
MPV_3000_24_LEFTGATTTCTCTCATAAATACAGAGATGTACAGCTT6
MPV_3000_24_RIGHTTCTCTAGAGGATATTCTCTCACATATAGATAATG6
MPV_3000_26_LEFTCATCTACCATTTCATTGATTTTAGCAGTGAA4
MPV_3000_26_RIGHTATCTATTTATAGCTCTTTGGATTCCTGATCT4
MPV_3000_28_LEFTAACAAAGTTATTCATCGTCGTCTACTATTCTAT8
MPV_3000_28_RIGHTCCTTACCATGAATAATATACATATCATACGGTA8
MPV_3000_30_LEFTAAGTGAGCAATTCCCAAGATTTCATCTA4
MPV_3000_30_RIGHTAGTTTTAACTTCTGAAACATCAATCTCCTTAC4
MPV_3000_32_LEFTCCTATTAGGACAGATACTACTATTTGTYTAAGT8
MPV_3000_32_RIGHTGTTTGTTAGAAGTCAGTCTAGTACTATTATAGT8
MPV_3000_34_LEFTTTACAGATGGAAGGGTCAAACTTAATAAAGG4
MPV_3000_34_RIGHTATGTATCTGATTGTACCATTTCATTGGGAA4
MPV_3000_36_LEFTGAGTATTGGGTAGAGTCTTACCTTACTATCTT4
MPV_3000_36_RIGHTCTAATTTTGTTTTGCTATCCGTTAATGATTTCC4
MPV_3000_38_LEFTTGTATTTCATTACCCAAGTTTGAGATTTCA6
MPV_3000_38_RIGHTGTGTTAATAACCCTACTGTTTCATTCTCCT6
MPV_3000_40_LEFTGATTGTGTTATATAACGGCGTTAATTATCT3
MPV_3000_40_RIGHTTTTGGTGATTCGAACGGTACACC3
MPV_3000_42_LEFTACGAGGGATTAGATGGTAAAATACAGAATAATAG3
MPV_3000_42_RIGHTGTCTTCACGAATTTCTATTTTCTTGGCC3
MPV_3000_44_LEFTGACAATTCTTTTGTATTCATAGACACTCGTTTT4
MPV_3000_44_RIGHTCGTAAGCGATAGTTGGTTGAAAGATAG4
MPV_3000_46_LEFTCCTTATCTTCATAGAATACTAAAGGCAATAAAG8
MPV_3000_46_RIGHTTAGAAAACCAGAAACTAATTACTACATTCATCC8
MPV_3000_48_LEFTCGACGTCAATTGTCTATAAATCAGAGTATAAAT6
MPV_3000_48_RIGHTATTTGGATCGTACGAAGATGTAGATAATAA6
MPV_3000_50_LEFTCCATCATTTCATTAATAGTATACGCAGAAAAGA6
MPV_3000_50_RIGHTCTTATTAACTTCKGTTGATTTATACGGAGTCT6
MPV_3000_52_LEFTTTGGATAATTCGCGCACCAACAA1.5
MPV_3000_52_RIGHTAGTGCCGGAATAAAATAAGGTGGATAC1.5
MPV_3000_54_LEFTCATACATCTGCTGCCAAAGTTATTTGC6
MPV_3000_54_RIGHTCTCTAATTCATCATGTGCYAACGTCT6
MPV_3000_56_LEFTATATTATCGGATTCGGTATTGTTACTCGAG3
MPV_3000_56_RIGHTAGGAGATAAGGAGGACCAACCAATTAA3
MPV_3000_58_LEFTTAGGATATGCTTTCATAAAGTCCCTAATAACTT6
MPV_3000_58_RIGHTACTCCATGATATGATAAATCCATGTAAAATAGC6
MPV_3000_60_LEFTmodCAACATTTATAGTATTTGTACAGCATACAGATC6
MPV_3000_60_RIGHTGCTTATACATCGATATCAGTTGTATTTTCTACT6
MPV_3000_62_LEFTGTGTACGTGCAACTGGTAATTAAAATAAAAAG4
MPV_3000_62_RIGHTGTAGTGTATATCATTAACTTCATTACGTATGAC4
MPV_3000_64_LEFTCTTTCCTAGATACTGCATATACTATCATAGATCA6
MPV_3000_64_RIGHTAGAAATCAAACAGGAGAATAATCAATGATGC6
MPV_3000_66_LEFTGATGGATGAGATATACAGCTATTAATTTCGAAA8
MPV_3000_66_RIGHTCTATGTTTAAMCGGTTCGCATTTATACATT8
MPV_3000_68_LEFTATAAAATGGATAACGGTACTCTAGAATTTACAC4
MPV_3000_68_RIGHTCGTCCATTTTCAAGCATTAGTCTTATACTATT4
MPV_3000_70_LEFTCATAAATTGCTACCGTGAATATAAATCCGTTA2
MPV_3000_70_RIGHTATGTCTCCCGCCTCTTGATCAC2
MPV_3000_72_LEFTGAAAGAATGTATTCATTTCTCCAGCGTC3
MPV_3000_72_RIGHTAGGATTAATGTTTAACCAGGATGTAATAACAC3
MPV_3000_74_LEFTCTAGTAGATTACGACATTAAACTCAAAACCAAT2
MPV_3000_74_RIGHTmodGTGTTGGGTACGACCGCTTATA2
MPV_3000_76_LEFTTTAACCGTGCGTAAAATGACTCGAG2
MPV_3000_76_RIGHTATAATCATCATGTTGAGGAAGTGGATTAC2
MPV_3000_78_LEFTGGTTGGTTAAAATCTAGTGGYTATGGG6
MPV_3000_78_RIGHTGTTTAGGACGGAACMCAAAAGGG6



Preparation of primer pools from individual primers
Preparation of primer pools from individual primers

Note
See Materials for primer sequences.


Combine separately the respective primer volumes listed in Table 1 and Table 2 using 100µM stocks to create the 10x Pool 1 and 10x Pool 2 primer pool stocks.
10x Pool 1: Combine the volumes listed in Table 1 for each of the 100µM 80 primers into a 1.7mL tube
10x Pool 2: Combine the volumes listed in Table 2 for each of the 100µM 78 primers into a 1.7mL tube
Pool 1 primerVol (µL)
MPV_3000_1_LEFT 4
MPV_3000_1_RIGHTmod 4
MPV_3000_3_LEFT 2
MPV_3000_3_RIGHT 2
MPV_3000_5_LEFTmod 2
MPV_3000_5_RIGHT 2
MPV_3000_7_LEFT 4
MPV_3000_7_RIGHT 4
MPV_3000_9_LEFT 8
MPV_3000_9_RIGHT 8
MPV_3000_11_LEFT 6
MPV_3000_11_RIGHTmod 6
MPV_3000_13_LEFT 6
MPV_3000_13_RIGHT 6
MPV_3000_15_LEFT 4
MPV_3000_15_RIGHT 4
MPV_3000_17_LEFT 8
MPV_3000_17_RIGHT 8
MPV_3000_19_LEFT 3
MPV_3000_19_RIGHT 3
MPV_3000_21_LEFT 3
MPV_3000_21_RIGHT 3
MPV_3000_23_LEFT 4
MPV_3000_23_RIGHT 4
MPV_3000_25_LEFT 4
MPV_3000_25_RIGHT 4
MPV_3000_27_LEFT 4
MPV_3000_27_RIGHT 4
MPV_3000_29_LEFT 6
MPV_3000_29_RIGHT 6
MPV_3000_31_LEFT 3
MPV_3000_31_RIGHT 3
MPV_3000_33_LEFT 3
MPV_3000_33_RIGHT 3
MPV_3000_35_LEFTmod 6
MPV_3000_35_RIGHT 6
MPV_3000_37_LEFT 6
MPV_3000_37_RIGHT 6
MPV_3000_39_LEFT 6
MPV_3000_39_RIGHT 6
MPV_3000_41_LEFT 6
MPV_3000_41_RIGHT 6
MPV_3000_43_LEFT 8
MPV_3000_43_RIGHT 8
MPV_3000_45_LEFT 4
MPV_3000_45_RIGHT 4
MPV_3000_47_LEFT 4
MPV_3000_47_RIGHT 4
MPV_3000_49_LEFT 4
MPV_3000_49_RIGHT 4
MPV_3000_51_LEFT 4
MPV_3000_51_RIGHT 4
MPV_3000_53_LEFT 12
MPV_3000_53_RIGHT 12
MPV_3000_55_LEFT 3
MPV_3000_55_RIGHT 3
MPV_3000_57_LEFT 6
MPV_3000_57_RIGHT 6
MPV_3000_59_LEFT 4
MPV_3000_59_RIGHT 4
MPV_3000_61_LEFT 4
MPV_3000_61_RIGHT 4
MPV_3000_63_LEFT 4
MPV_3000_63_RIGHT 4
MPV_3000_65_LEFT 6
MPV_3000_65_RIGHT 6
MPV_3000_67_LEFT 8
MPV_3000_67_RIGHT 8
MPV_3000_69_LEFT 4
MPV_3000_69_RIGHT 4
MPV_3000_71_LEFTmod 4
MPV_3000_71_RIGHTmod 4
MPV_3000_73_LEFTmod 4
MPV_3000_73_RIGHT 4
MPV_3000_75_LEFT 3.5
MPV_3000_75_RIGHT 3.5
MPV_3000_77_LEFT 4
MPV_3000_77_RIGHT 4
MPV_3000_79_LEFT 12
MPV_3000_79_RIGHT 12
Table 1 - 10x Pool 1 of 2500bp V2.3.4 amplicon primer scheme
Pool 2 primerVol (µL)
MPV_3000_2_LEFT 4
MPV_3000_2_RIGHT 4
MPV_3000_4_LEFT 3
MPV_3000_4_RIGHT 3
MPV_3000_6_LEFT 3
MPV_3000_6_RIGHT 3
MPV_3000_8_LEFT 4
MPV_3000_8_RIGHT 4
MPV_3000_10_LEFT 4
MPV_3000_10_RIGHTmod 4
MPV_3000_12_LEFT 2
MPV_3000_12_RIGHT 2
MPV_3000_14_LEFT 3
MPV_3000_14_RIGHT 3
MPV_3000_16_LEFT 6
MPV_3000_16_RIGHT 6
MPV_3000_18_LEFT 4
MPV_3000_18_RIGHT 4
MPV_3000_20_LEFT 2
MPV_3000_20_RIGHT 2
MPV_3000_22_LEFT 3
MPV_3000_22_RIGHT 3
MPV_3000_24_LEFT 6
MPV_3000_24_RIGHT 6
MPV_3000_26_LEFT 4
MPV_3000_26_RIGHT 4
MPV_3000_28_LEFT 8
MPV_3000_28_RIGHT 8
MPV_3000_30_LEFT 4
MPV_3000_30_RIGHT 4
MPV_3000_32_LEFT 8
MPV_3000_32_RIGHT 8
MPV_3000_34_LEFT 4
MPV_3000_34_RIGHT 4
MPV_3000_36_LEFT 4
MPV_3000_36_RIGHT 4
MPV_3000_38_LEFT 6
MPV_3000_38_RIGHT 6
MPV_3000_40_LEFT 3
MPV_3000_40_RIGHT 3
MPV_3000_42_LEFT 3
MPV_3000_42_RIGHT 3
MPV_3000_44_LEFT 4
MPV_3000_44_RIGHT 4
MPV_3000_46_LEFT 8
MPV_3000_46_RIGHT 8
MPV_3000_48_LEFT 6
MPV_3000_48_RIGHT 6
MPV_3000_50_LEFT 6
MPV_3000_50_RIGHT 6
MPV_3000_52_LEFT 1.5
MPV_3000_52_RIGHT 1.5
MPV_3000_54_LEFT 6
MPV_3000_54_RIGHT 6
MPV_3000_56_LEFT 3
MPV_3000_56_RIGHT 3
MPV_3000_58_LEFT 6
MPV_3000_58_RIGHT 6
MPV_3000_60_LEFTmod 6
MPV_3000_60_RIGHT 6
MPV_3000_62_LEFT 4
MPV_3000_62_RIGHT 4
MPV_3000_64_LEFT 6
MPV_3000_64_RIGHT 6
MPV_3000_66_LEFT 8
MPV_3000_66_RIGHT 8
MPV_3000_68_LEFT 4
MPV_3000_68_RIGHT 4
MPV_3000_70_LEFT 2
MPV_3000_70_RIGHT 2
MPV_3000_72_LEFT 3
MPV_3000_72_RIGHT 3
MPV_3000_74_LEFT 2
MPV_3000_74_RIGHTmod 2
MPV_3000_76_LEFT 2
MPV_3000_76_RIGHT 2
MPV_3000_78_LEFT 6
MPV_3000_78_RIGHT 6
Table 2 - 10x Pool 2 of 2500bp V2.3.4 amplicon primer scheme
Create 10µM (1x) stock solutions of pool 1 and pool 2 of an appropriate volume in individual 1.7mL tubes from the 100µM stocks created above by dilution in Ultrapure water. Primers at this concentration should only be freeze/thawed a maximum of three times.
Preparation of PCR Reagents
Preparation of PCR Reagents
Prepare both PCR Master Mix 1 (MM1 containing Primer Pool 1) and 2 (MM2 containing Primer Pool 2) separately in a PCR Clean Room as follows:
ReagentVolume (Rxn)Volume (24 Rxns)
Ultrapure water 15.65µl 375.6µl
5X Q5 Reaction Buffer 5µl 120µl
10mM dNTPs 0.5µl 12µl
Q5 Hot Start DNA Polymerase 0.25µl 6µl
Primer Pool 1 or 2 1.1µl 26.4µl
Total 22.5µl 540µl
Label two PCR Plates with the experiment code followed by “PCR Pool 1” or “PCR Pool 2.” Using a repeater pipet, pipet 22.5µl of PCR MM1 into “PCR Pool 1” and 22.5µl of PCR MM2 into “PCR Pool 2.”
Seal and move all the plates containing your master mixes from the Clean Reagent Preparation Room into a Genomic Level Room.
Generate PCR amplicons
Generate PCR amplicons

Note
Sample template material
Sample nucleic acid extracts previously found to be positive for Mpox virus by a validated laboratory method are used. At the BCCDC PHL this Mpox DNA template material is generated using the 5X MagMax - 96 Viral Isolation kit from Applied Biosystems from preferred higher yielding clinical sample site swabs, such as lesions and mucosal sources, using a Copan swab with Universal Transport Media.

Pipet 2.5µl of template into their respective wells in PCR Pool 1 plate. Pipet to mix.
Pipet 2.5µl of template into their respective wells in PCR Pool 2 plate. Pipet to mix.
Seal the two PCR plates with an adhesive seal and spin down.
Place the two PCR plates on thermal cyclers and run at the following conditions:

ABC
Temperature Time Cycles
98°C30 seconds 1
98°C15 seconds 35
65°C5 minutes
4°C∞1
Thermal cycling program for Mpox amplicon generation. This program takes 3.5 hours. PCR amplicons are stable for several days at 4°C and months at -20°C
Amplicons from primer pools 1 and 2 are combined post-PCR for NGS library preparation.
Sequencing
Sequencing
Proceed with library preparation and sequencing of Mpox amplicons. A condensed library preparation protocol for the Illumina NextSeq may be used for Mpox amplicons as per Hickman et al (2024). Protocols.io link here. For other NGS platforms proceed with appropriate methods and analysis.
CITATION
Hickman R, Nguyen J, Lee TD, Tyson JR, Azana R, Tsang F, Hoang L, Prystajecky NA (2024). Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit..
LINK
https://doi.org/10.1128/jcm.00103-22

Bioinformatics
Bioinformatics
For consensus building and variant calling from Illumina DNA Prep generated data see the BCCDC Nextflow pipeline here that uses a modified ARTIC network fieldbioinformatics tool for Mpxv created by the Simpson lab here.

For primer scheme .bed, primer_pair.tsv and associated reference sequences files see here

We use the below files for compatibility and integration with the publicly available Mpox Nextstrain instances using the same reference sequence NC_063383.1 :-

NC_063383.1_masked_right_itr.fasta (Mpox reference sequences with right ITR region masked)

NC_063383.1_masked_right_itr.fasta.fai (Index file for above mpox reference fasta file)

BCCDC-PHL_mpxv_v2.3_primer_pairs.tsv (File containing bracketing primer pairs for the generated amplicon regions. This is specific to the Illumina analysis pipeline we use and is for short read quality filtering when using library preps that fragment amplicons)

BCCDC-PHL_mpxv_v2.3_NC_063383.1.scheme.bed (.bed file containing primer binding site information for a specific reference for use in analysis pipelines)

BCCDC-PHL_mpxv_v2.3_NC_063383.1.primer.bed (.bed file containing individual primer reference location, name, pool number, orientation and sequence information **not compatible with analysis pipeline but useful to have information all together**)

Protocol references
Hickman R, Nguyen J, Lee TD, et al. Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit. J Clin Microbiol. 2024;62(3):e0010322. doi:10.1128/jcm.00103-22

GitHub - BCCDC-PHL/mpxv-artic-nf

Primer .bed, primer_pair.tsv and references sequences used

GitHub - BCCDC-PHL/mpxv-qc: Companion pipeline to perform quality control for BCCDC-PHL/mpxv-artic-nf