xGen™ Monkeypox Virus Amplicon Panel
xGen Amplicon Technology uses overlapping primer sets to provide comprehensive coverage of positions 6760–190,905 (ITRs not included) from as few as 300 monkeypox viral genome copies. Track the spread in a single-tube, ~2.5-hour workflow.
xGen NGS—made for monkeypox research.
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Monkeypox has been termed a global health emergency. For researchers interested in tracking the evolution of the monkeypox virus genome, it can be difficult to get sufficient coverage. The xGen Monkeypox Virus Amplicon Panel provides a single-tube, two-step PCR amplification workflow with primer sets designed to create amplicons across the entire genome.*
The xGen Monkeypox Virus Amplicon Panel supports:
- Comprehensive coverage from positions 6760–190,905 (ITRs not included)
- Sequence data from viral titers as low as 300 viral genome copies
- Single-tube workflow
- Viral DNA-to-sequencer in ~2.5 hours
- Up to 1536 UDIs
- Super amplicon technology
*See Product Data section for more information. Panel design omits ITRs due to the highly repetitive nature of these sequences.
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IDT is committed to providing quality products to researchers working on the cutting edge of scientific discovery. The xGen Monkeypox Virus Amplicon Panel was designed as part of the next generation sequencing (NGS) Tech Access program at IDT, which is intended to accelerate innovation by enabling earlier access to our most advanced research tools. Tech Access products have not been through our standard, rigorous development cycle. These products are particularly well suited for researchers who require the most up-to-date technology to unlock new discoveries.
The current variants of monkeypox that are circulating have a genome of nearly 200 kb double-stranded DNA [1]. Surveillance of the virus and any potential mutations have gained international support due to the lessons learned through the COVID-19 pandemic. The IDT xGen Monkeypox Virus Amplicon Panel helps to enable researchers to track monkeypox strains, including potential new variants, by next generation sequencing.
DNA-to-sequencer in 2.5 hours
The workflow for the xGen Monkeypox Virus Amplicon Panel starts with extracted viral DNA (Figure 1). You can then generate an NGS library in a single tube using tiled primer pairs designed to target 184 kb of the monkeypox genome. Primers were designed for the currently circulating strain of the monkeypox virus (NCBI accession number ON568298 [1]), which allows you to generate overlapping amplicons in a single-tube, PCR 1 + PCR 2 workflow. If pooling multiple samples for NGS, the xGen Amplicon Core Kit includes the reagents for Normalase™ technology, the proprietary enzymatic normalization step that reduces hands-on time needed for manual normalization. Specifications of the Tech Access, xGen Monkeypox Virus Amplicon Panel are found in Table 1.
Figure 1. xGen Monkeypox Virus Amplicon Panel workflow. A dual-index library is prepared from viral samples in three main steps: 1) multiplex PCR, 2) adapter attachment with indexing PCR, and 3) an optional Normalase™ step to produce equimolar library pools.
Table 1. Features of the xGen Monkeypox Virus Panel.
| Features | Specifications |
|---|---|
| Design coverage and panel information |
|
| Input Material |
|
| Time | ~2.5 hours for viral DNA-to-library |
| Multiplexing capability | Up to 1536 UDIs |
| Compatible with other indexes? | Yes |
| Recommended depth | Strain identification or variant calling: 500K reads per library |
Product data
The recent emergence of monkeypox viral infections globally has resulted in an increased need for rapid, reliable NGS approaches to not only monitor and trace outbreaks but to also track any potentially novel variants that may arise. Epidemiological studies are currently underway to pinpoint transmission and infection patterns of this zoonotic disease. IDT recognizes the importance of these studies and has designed an xGen NGS Amplicon Sequencing Panel to target the monkeypox virus†.
The xGen Monkeypox Virus Amplicon Panel offers a streamlined (DNA-to-sequencer in 2.5 hours), single-tube NGS workflow for studying the monkeypox virus (MPXV). This Predesigned xGen Amplicon Panel provides 184 kb of high-quality coverage of the monkeypox genome (Table 2, Figure 2 and Figure 3) from inputs as low as 300 viral genome copies (Table 3). xGen Amplicon technology includes amplicon tiling and creation of super amplicons to ensure comprehensive genome coverage and provide resistance to future viral mutations that may fall on a priming site (Figure 4), thus enabling future identification of novel variants.
Figure 2. Coverage of the monkeypox virus genome. As an example of comprehensive coverage, at the specified positions, of monkeypox (DQ011157), ~3000 copies of the monkeypox genome (BEI Resources, NIAID, NIH: Genomic DNA from Monkeypox Virus, USA-2003, NR-4928) and 10 ng Coriell DNA NA12878 (human) were input into the xGen Amplicon workflow using the xGen Monkeypox Virus Amplicon Panel. The resulting NGS library was sequenced on a MiSeq system (Illumina) with 250 bp paired-end (PE) sequencing with 3,055,632 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). The total sequencing depth per genomic position (in log10 scale) was then plotted to visualize genomic coverage. Example data for one replicate are shown here, though experimental duplicates showed similar results.
Coverage and on-target mapping rates
Based on initial research and development, this panel has been shown to offer comprehensive coverage of the monkeypox viral genome from positions 6760–190,905. The inverted terminal repeats (ITRs) at both ends of the genome were omitted from the panel design due to the repetitive nature of these sequences.
To prepare amplicon sequencing libraries using the xGen Monkeypox Virus Amplicon Panel, ~3000 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human) were used. The resulting NGS library was sequenced on a MiniSeq™ system (Illumina) with 150 bp paired-end (PE) sequencing with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). Table 2 shows representative metrics obtained in this proof-of-concept experiment.
Table 2. xGen Monkeypox Virus Amplicon Panel NGS metrics.
| % mapping | % on-target (base) | % base uniformity (>0.2X mean) | |
|---|---|---|---|
| xGen Monkeypox Virus Amplicon Panel | 88.1 | 97.7 | 98.0 |
Figure 3. The xGen Monkeypox Virus Amplicon Panel results in comprehensive coverage of the monkeypox genome (excluding the ITRs). To prepare amplicon sequencing libraries using the xGen Monkeypox Virus Amplicon Panel, ~3000 copies of the monkeypox genome (BEI Resources, NIAID, NIH: Genomic DNA from Monkeypox Virus, USA-2003, NR-4928) and 10 ng Coriell DNA NA12878 (human) were used. The resulting NGS library was sequenced on a MiniSeq system (Illumina) with 150 bp paired-end (PE) sequencing with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). The shown dot plot indicates the relative coverage of each amplicon in the panel (n = 4), where each amplicon is represented by a blue dot. Colored dashed lines represent mean coverage of 0.05X, 0.1X, 0.2X, 1X, and 5X.
Sequencing results from titers as low as 300 viral genome copies
The input into xGen Monkeypox Virus Amplicon Panel consisted of either 3000, 300, or 0 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human). The resulting NGS library was sequenced as described above. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]). A high level of genomic coverage was observed with 3000 and 300 monkeypox genomic copies (Table 3), and no genomic coverage was observed with no monkeypox copy input.
Table 3. The xGen Monkeypox Virus Amplicon Panel provides coverage for a range of viral DNA inputs.
| Sample number | Input copies of viral genomes | Total reads | Percent target bases 10X (base) |
|---|---|---|---|
| 1 | 3000 | 2,374,500 | 99.4 |
| 2 | 2,725,540 | 99.4 | |
| 3 | 300 | 1,984,616 | 98.8 |
| 4 | 1,963,414 | 98.7 | |
| 5 | 0/NTC | 2,183,412 | 0.2 |
| 6 | 2,464,906 | 0.3 |
Super amplicons help maintain sequencing coverage despite mutations
Figure 4. The xGen Monkeypox Virus Amplicon Panel maintains genomic coverage despite mutations at primer binding sites. The generation of super amplicons means that even in the case of a mutation occurring in primer binding sites (shown here by black arrows), the xGen Monkeypox Virus Amplicon Panel can maintain genomic coverage. The input into library prep consisted of ~3000 copies of the monkeypox genome (BEI Resources) and 10 ng Coriell DNA NA12878 (human). The resulting NGS library was sequenced on a MiniSeq system (Illumina) (150 bp PE sequencing) with 1,774,058 total reads. Reads were aligned and mapped to the monkeypox reference genome (DQ011157 [2]) using bwa (v 2.2.1 [3]) and coverage was visualized with IGV (Broad Institute [4]).
Resources
References
- Antwerpen MH, Lang D, Zange S, et al. First German genome sequence of Monkeypox virus associated to multi-country outbreak in May 2022. 2022.
- Likos AM, Sammons SA, Olson VA, et al. A tale of two clades: monkeypox viruses. J Gen Virol. 2005;86(Pt 10):2661-2672.
- Li H. and Durbin R. Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics. 2009; 25:1754-60.
- Robinson JT, Thorvaldsdottir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol. Jan 2011;29(1):24-6. doi:10.1038/nbt.1754
†The xGen Monkeypox Virus Amplicon Panel was designed as part of the next generation sequencing (NGS) Tech Access program at IDT, which is intended to accelerate innovation by enabling earlier access to our most advanced research tools. Tech Access products have not been through our standard, rigorous development cycle.
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