Dec 13, 2022
Version 4
Monkeypox virus multiplexed PCR amplicon sequencing (PrimalSeq) V.4
- Nicholas F.G. Chen*1,
- Luc Gagne*2,
- Matthew Doucette2,
- Sandra Smole2,
- Erika Buzby2,
- Joshua Hall2,
- Stephanie Ash2,
- Rachel Harrington2,
- Seana Cofsky2,
- Selina Clancy2,
- Curtis J Kapsak3,
- Joel Sevinsky3,
- Kevin Libuit3,
- Mallery I Breban1,
- Chrispin Chaguza1,
- Nathan D. Grubaugh1,
- Daniel J. Park4,
- Glen R. Gallagher#2,
- Chantal B.F. Vogels#1
- 1Department of Epidemiology of Microbial Diseases, Yale School of Public Health;
- 2Massachusetts Department of Public Health;
- 3Theiagen Genomics;
- 4Broad Institute, Cambridge, Massachusetts
Protocol Citation: Nicholas F.G. Chen*, Luc Gagne*, Matthew Doucette, Sandra Smole, Erika Buzby, Joshua Hall, Stephanie Ash, Rachel Harrington, Seana Cofsky, Selina Clancy, Curtis J Kapsak, Joel Sevinsky, Kevin Libuit, Mallery I Breban, Chrispin Chaguza, Nathan D. Grubaugh, Daniel J. Park, Glen R. Gallagher#, Chantal B.F. Vogels# 2022. Monkeypox virus multiplexed PCR amplicon sequencing (PrimalSeq) . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvob1nbl4o/v4Version created by Nathan D Grubaugh
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: December 12, 2022
Last Modified: February 11, 2023
Protocol Integer ID: 73877
Keywords: Monkeypox, MPXV, Amplicon sequencing, PrimalSeq, hMPXV
Disclaimer
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Abstract
Version 4 Updates:
- Added a fork to the primer preparation step for use with pre-pooled primers (Yale hMPXV Amplicon Panel) from IDT
Background: The current global outbreak of human Monkeypox virus (hMPXV) concurrent with an ongoing SARS-CoV-2 pandemic has further highlighted the need for genomic surveillance and pathogen whole genome sequencing. While metagenomic and hybrid capture sequencing approaches were used to sequence many of the early mpox cases, the viability of these methods is dependent on samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the current outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there is a strong need for a more sensitive and broadly applicable sequencing approach. Amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to design a primer scheme for hMPXV and we validated it with widely used SARS-CoV-2 sequencing protocols. Based on initial validation, our approach shows notably higher depth and breadth of coverage across the genome, particularly with higher PCR cycle threshold (Ct) samples, as compared to metagenomic sequencing. While further testing is needed, the early success of this approach has significant implications for sequencing efforts of the current hMPXV outbreak and serves as a proof of concept of amplicon-based sequencing for use with other large-genome DNA viruses and potentially bacterial genomes.
Overview of Design: We used PrimalScheme (https://primalscheme.com/) to generate an hMPXV primer scheme using a pre-outbreak A.1 lineage reference genome (GenBank accession: MT903345). The primer scheme comprises a total of 163 primer pairs with an amplicon length ranging between 1597 and 2497 bp (average length of 1977 bp). The primer scheme is compatible with current ARTIC and COVIDSeq SARS-CoV-2 sequencing protocols, and while validated only with Illumina library prep methods and sequencing platforms, it would likely see a high degree of success with other sequencing platforms such as the Oxford Nanopore Technologies MinION.
Initial Validation: We validated our hMPXV primer scheme with clinical specimens at the Massachusetts Department of Public Health, Massachusetts State Public Health Laboratory under the IRB Project Titled “Rash Illness: Alternate Specimen Types and Sequencing” (protocol number 1917413). A total of 25 clinical specimens were included in this initial validation comprised of both throat and swabs of fluid from lesions of 8 individuals, belonging to the current outbreak-associated hMPXV B.1 lineage as determined by prior characterization at the Centers of Disease Control and Prevention. Ct values were determined with the non-variola orthopox diagnostic qPCR assay developed for use by the Laboratory Response Network (Rapid Diagnostic Testing for Response to the Monkeypox Outbreak — Laboratory Response Network, United States, May 17–June 30, 2022 | MMWR (cdc.gov)). Clinical samples ranged in cycle threshold (Ct) values from 15.03 (high viral concentration) to 34.63 (low viral concentration), and each sample was sequenced in parallel using a metagenomics approach and the provided amplicon-based approach. Extractions and sequencing analysis were performed in accordance with current biosafety guidance including extraction in a BSL-3 setting. An evaluation of site-specific biosafety practices should be developed in consultation with your organization’s biosafety officer. Libraries were prepared with the Illumina DNA prep kit and sequenced on the MiSeq (v2 kit running 2x150 nt reads). Consensus genomes were generated at 10X coverage using the TheiaCoV_Illumina_PE Workflow Series on Terra.bio. An hMPXV “fork” was developed for us by Curtis Kapsak from Theiagen, which included the hMPXV reference sequence, primer scheme, and consensus genome length, and can currently be accessed at: https://github.com/theiagen/public_health_viral_genomics/tree/cjk-MPXV-theiacov. We found comparable genome coverage between amplicon and metagenomic sequencing with low Ct (<18) samples, yet a significant increase in genome coverage with amplicon sequencing in higher Ct samples (>18; Figure 1A.) The library clean up stage of the amplicon based Illumina DNA prep protocol was conducted using the ‘standard DNA input’ option for comparison to metagenomics. Following optimization with 15 additional paired samples unrelated to the initial 10 samples, we found improved genome coverage with higher Ct samples (>25) using the ‘small PCR amplicon input’ option of the Illumina DNA prep protocol (Figure 1B.) Our findings highlight how amplicon-based approaches can significantly expand hMPXV sequencing to a wider variety of samples.
Amongst samples with a low Ct (<18), genome coverage via amplicon sequencing was consistently >97%, with minimal amplicon drop-outs (Figure 2). Amplicons 11, 75, and 118 showed consistent drop-out across the sequenced samples, while none of the primers had mismatches, except for a single nucleotide mismatch in the 11_RIGHT primer. We did obtain coverage for these amplicons when sequencing clade IIa DNA from cultured virus (strain USA-2003; NR-4928) obtained from BEI Resources (NIAID, NIH). This suggests that the dropouts of these primers may be a specific issue related to the current hMPXV B.1 lineage genomes. As this protocol is still in development, we will further investigate performance as we continue to sequence additional samples.
Conclusion: We developed an amplicon-based sequencing (PrimalSeq) approach for hMPXV that improved the depth and breadth of genome coverage with low viral concentration specimens as compared to metagenomic sequencing.
This protocol represents the fourth iteration of development. Further versions will be uploaded to protocols.io with an accompanying description of changes as appropriate.
Materials
MPXV Primer Scheme
Primers may be ordered from any oligonucleotide company using the following file as a reference. For instructions on how to prepare hMPXV Primer Pool 1 and 2 (10 uM) see the "Unpooled Primers" fork of step 1 in the protocol.
MPXV-primer_genome-positions.tsv Our pre-pooled primer scheme can also be ordered through Integrated DNA Technologies (IDT) with the following link. For preparation instructions, follow the "IDT Pre-Pooled Primers" fork of step 1 in the protocol.
Library Preparation Method: Illumina DNA Prep
Library Preparation Method: CovidSeq
| A | B | C | |
| Illumina COVIDSeq Test Box 1 – 3072 Samples, Part # 20044408 | |||
| Reagent | Description | Storage | |
| ITB | Illumina Tune Beads | Room Temperature | |
| ST2 HT | Stop Tagment Buffer 2 HT | Room temperature, post-amp environment | |
| Illumina COVIDSeqTest Box 2 – 3072 Samples, Part # 20044409 | |||
| EBLTS HT | Enrichment BLT HT | 2°C to 8°C post-amp environment | |
| TWB HT | Tagmentation Wash Buffer HT | 2°C to 8°C post-amp environment | |
| RSB HT | Resuspension Buffer HT | 2°C to 8°C, post-amp environment | |
| Illumina COVIDSeq Test Box 3 – 3072 Samples, Part # 20044410 | |||
| IPM HT | Enhanced PCR Mix HT | -25°C to -15°C, pre-amp environment | |
| TB1 HT | Tagmentation Buffer 1 HT | -25°C to -15°C, post-amp environment | |
| EPM HT | Enhanced PCR Mix HT | -25°C to -15°C, pre-amp environment | |
| Index Adapater Part Numbers : 20043132, 20043133, 20043134, 20043135 | |||
| Index Adapters | IDT for Illumina- PCR Indexes Set 1-4 | Room Temperature | |
Additional Materials
| A | B | C | |
| Reagent | Description | Storage | |
| 80% EtOH | 80% Ethanol | Room Temperature | |
| Nuclease-free water | Room Temperature |
Safety warnings
Processing of any sample type which could potentially be positive for hMPXV should be conducted in BSL2+ settings. Before starting work with these samples, please contact your local EHS (environment, health and safety) or biosafety office for proper guidance on how to work with these samples in your laboratory.
Before start
This protocol is currently in the developmental phase. It is for research purposes only and should not be used in a diagnostic capacity.
If using clinical samples, DNA will need to be extracted and purified prior to beginning library preparation.
Primer diluting and pooling requires several hours and may be conducted well in advance so long as the pooled primers are stored at -20°C.
Following primer dilution and pooling, this workflow can be completed in one day, however, it is recommended to be conducted across two, with the amplicon generation step on the first day and all subsequent wet-lab steps on the second day.
Protocol Recommendations:
To maximize sequencing coverage, we recommend the following best practices based on our multi-site validation study:
- Prioritizing samples with cycle threshold (Ct) values <29 if resources are limited
- Generating at least 1 million sequencing reads per sample
Primer Preparation
Primer Preparation
Primer Source
Dilute primers to a working concentration of 10uM with nuclease-free water (i.e. 1:10 dilution)
IDT Pre-Pooled PrimersFrom 56 to 81 steps
Integrated DNA Technologies (IDT) Pre-Pooled Yale hMPXV Amplicon Panel:
*Safe Stopping Point: Pooled Primers Can Be Stored at -20°C*
Library Preparation Method
Amplicon Generation
Amplicon Generation
Reagents:
Illumina CovidSeq Test (RUO)53 steps
In two separate tubes, prepare the following master mixes:
Note
Master mix volumes are for one reaction and do not account for lost volume due to pipetting. Multiply volumes by reaction number accordingly.
Label two sets of PCR tubes/plates for Pool 1 and Pool 2
Add the following:
- 20 µL Pool 1 master mix to each Pool 1 tube/well
- 20 µL Pool 2 master mix to each Pool 2 tube/well
Add 5 µL DNA to each tube in both sets
- Mix by pipetting up and down 10 times
- Briefly centrifuge tubes/plates
Note
Be sure to include a negative PCR control (NTC; nuclease-free water) for each pool
Place on thermocycler and run the following program (choose preheat lid option):
*Safe Stopping Point: Amplified DNA may remain in the thermocycler at 4°C or stored at -20°C until ready to use*
Amplicon Tagmentation and Clean Up
Amplicon Tagmentation and Clean Up
Reagents:
Spin down PCR tubes/plates
Prepare the following master mix:
In each PCR tube add:
- 30 µL Master mix
- 10 µL Pool 1 amplicons
- 10 µL Pool 2 amplicons
For a total reaction volume of 50 µL per PCR tube/well
Note
Pooling of amplicons should be conducted on a dedicated post-PCR bench to prevent contamination
Mix by pipetting up and down and briefly centrifuge
Place on thermocycler and run the following program (choose preheat lid option):
Once the thermocycler reaches 10 °C , remove tubes/plates and spin down
Add 10 µL ST2 to each tube/well, mix by pipetting up and down, and briefly centrifuge
Incubate at Room temperature for 00:05:00
5m
Place on magnetic stand and wait until liquid is clear (a few minutes)
Remove and discard all supernatant
Remove tubes/plates from magnetic stand and add 100 µL TWB to each tube/well
- Mix by pipetting up and down 10 times and spin down. Be careful to not introduce bubbles.
Repeat steps 4.9 - 4.11. Leave the supernatant after the second wash so that the beads don’t dry out
Amplify Tagmented Amplicons
Amplify Tagmented Amplicons
Reagents:
Prepare the following master mix:
Place the tubes/plates with tagmented amplicons on the magnetic stand and remove the supernatant once the liquid is clear
Use a 20µL pipette to remove any residual TWB from tubes/wells
Remove the tubes/plates from the magnetic stand
Add 40 µL master mix to each tube/well
Add 10 µL dual-barcoded index adapters to each tube/well
Note
Note which set of indexes are used for each tube for bioinformatic processing
Pipette up and down to mix and spin down
Place on thermocycler and run the following program (choose preheat lid option):
Pool and Clean Up
Pool and Clean Up
Reagents:
Briefly centrifuge tubes/plates
Place on magnetic stand and wait until liquid is clear (a few minutes)
Pool libraries by equal volume:
Pipette up and down to mix pooled libraries and briefly spin down
Calculate the volume of ITB to reach a 0.6X beads:total pool volume ratio
Note
For example: Total pooled volume is 400 µL (10 samples x 40 µL/sample)
then add 0.6 x 400 µL = 240 µL ITB
Mix by pipetting up and down, briefly centrifuge, and incubate at Room temperature for 00:05:00
5m
Place on magnetic stand and wait until liquid is clear (a few minutes)
Transfer supernatant to a new tube (do not discard, this is your final library). This volume will be slightly lower than the total combined volume
Note
For example: 400µL+ 240µL = total 640µL > transfer 630µL
Calculate the volume of beads to add to the supernatant to attain a second clean-up beads:sample ratio of 0.9X:
Vo = total volume of sample + beads from step 5.5
Vt = transferred volume of supernatant
Note
For example: 630µL of supernatant is transferred to a new tube
Add 118µL of ITB to 630µL of transferred supernatant
Add beads to supernatant, mix by pipetting up and down, briefly centrifuge, and incubate at Room temperature for 00:05:00
5m
Place on magnetic stand and wait until liquid is clear (a few minutes)
Carefully discard supernatant
Add 1000 µL 80% EtOH
Wait 00:00:30
30s
Repeat steps 6.12 - 6.14
Remove supernatant using a 20µL pipette to remove all residual EtOH
Add 55 µL of RSB to the tube, mix by pipetting up and down, and briefly centrifuge
Incubate at Room temperature for 00:02:00
2m
Place on magnetic stand and wait until liquid is clear (a few minutes)
Transfer 50 µL of final pooled library to a new 1.5mL tube
Quantify library on a Qubit and obtain fragment distribution using a Bioanalyzer/Tape Station
Note
Qubit SOP: Agilent High Sensitivity DNA Kit Guide.pdf
Agilent High Sensitivity DNA Kit Guide.pdf Bioanalyzer SOP: Agilent High Sensitivity DNA Kit Guide.pdf
Agilent High Sensitivity DNA Kit Guide.pdf Sequencing
Sequencing
Protocol validated on the MiSeq (v2 kit running 2x150 nt reads)
Note
Note: For sequencing we recommend generating at least 1 million reads per sample for optimal sequencing coverage
Sequencing may be performed on Illumina and Oxford Nanopore Technologies sequencing platforms following standard protocols
Bioinformatics/Analysis
Bioinformatics/Analysis
Sequencing results may be analyzed utilizing a standard amplicon sequencing bioinformatics pipeline, including those employed for SARS-CoV-2 sequencing.
Note
If utilizing a SARS-CoV-2 based bioinformatics pipeline, adjustments may be required to account for the consensus genome length
Reference Sequence: MPXV.reference.fasta
MPXV.reference.fasta Bed file: MPXV.primer.bed
MPXV.primer.bed