TITLE: BCCDC / ARTIC Mpox V2.3.4 2500bp amplicon generation and NGS
AUTHORS: 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

[DESCRIPTION]
  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.  


 


[STEPS]
SECTION: Preparation of primer pools from individual primers
1. 
 


SECTION: Preparation of PCR Reagents
2. 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:
 
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.” 
SECTION: Preparation of PCR Reagents
3. Seal and move all the plates containing your master mixes from the Clean Reagent Preparation Room
into a Genomic Level Room.
SECTION: Generate PCR amplicons
5. 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. 
SECTION: Generate PCR amplicons
6. Place the two PCR plates on thermal cyclers and run at the following conditions:

 
 
 Amplicons from primer pools 1 and 2 are combined post-PCR for NGS library preparation. 
SECTION: Preparation of primer pools from individual primers
1.1. 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.   
SECTION: Preparation of primer pools from individual primers
1.2. 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 
 
 
 
 
 
 
SECTION: Sequencing
7. 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.
 

SECTION: Bioinformatics
8. 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**)

SECTION: Bioinformatics
9. For QC analysis see the companion pipeline for quality control analysis.  
SECTION: Preparation of primer pools from individual primers
1.3. 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. 
SECTION: Generate PCR amplicons
4.