- 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.
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These products are particularly well suited for researchers who require the most up-to-date technology to unlock new discoveries.
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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.
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The IDT xGen Monkeypox Virus Amplicon Panel helps to enable researchers to track monkeypox strains, including potential new variants, by next generation sequencing.
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DNA-to-sequencer in 2.5 hours
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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.
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Specifications of the Tech Access, xGen Monkeypox Virus Amplicon Panel are found in Table 1.
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Please note that the term amplicons in the product refers to the same concept as the baits I mentioned in my talk.
- Table 1. Features of the xGen Monkeypox Virus Panel.
- Features Specifications
- Design coverage and panel information Comprehensive coverage from positions 6760–190,905. (ITRs not included); 1892 amplicons, sized 93–246 bp (average size is 150 bp)
- Input Material Extracted viral DNA; Suggested minimum of 300 viral genome copies
- Time ~2.5 hours for viral DNA-to-library
- Multiplexing capability Up to 1536 UDIs
- Compatible with other indexes? Yes
- Recommended depth
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Strain identification or variant calling: 500K reads per library. However, we have 70K(70896)x2 reads in the sample Affe31!
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UDI stands for Unique Dual Index. This refers to a method used in next-generation sequencing (NGS) where each sample is tagged with a unique combination of two indices (dual indices) to enable multiplexing, i.e., the simultaneous sequencing of multiple samples in a single run. The dual indexing helps to distinguish different samples and minimizes the risk of index hopping or misassignments.
- Index hopping（索引跳跃） 是下一代测序（Next-Generation Sequencing, NGS）中的一种现象。在这种情况下，用于标识不同样本的索引（条形码）会意外地在样本之间“跳跃”。这意味着某个样本的索引对（双索引）可能会与其他样本混合，从而导致测序数据错误地分配。具体来说，索引跳跃会导致特定样本的序列与错误的条形码关联。例如，如果样本A的索引与样本B的序列混淆，最终的分析可能会错误地将样本B的序列归类为样本A的序列。这种现象在使用某些测序平台（如Illumina NovaSeq）时尤其明显，因为在聚类或扩增过程中，适配子分子可能会交换条形码。为降低索引跳跃的风险，研究人员可以使用双重索引（即使用两个索引）和特定的文库准备方法。
- 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.
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IDT recognizes the importance of these studies and has designed an xGen NGS Amplicon Sequencing Panel to target the monkeypox virus†.
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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).
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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.
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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. !!
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proprietary 英 [prəˈpraɪətri] adj. 专有的，专利的; 所有（人）的; （商品）专卖的
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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.
- name | % mapping | % on-target (base) | % base uniformity (>0.2X mean)
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xGen Monkeypox Virus Amplicon Panel | 88.1 | 97.7 | 98.0
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Sequencing results from titers (浓度测定，滴定量) 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]).
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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.
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Table 3. The xGen Monkeypox Virus Amplicon Panel provides coverage for a range of viral DNA inputs.
- 他们的depth是 1-2 million reads, 但是你的depth只有70K!
- 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
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6 2,464,906 0.3
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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]).
- Since early May 2022, dozens of suspected and confirmed monkeypox infections have been reported in several European and North American countries.
- The first German monkeypox case was reported in Munich on May 19, where the 26 year-old patient had shown characteristic skin changes.
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During his travel through Europe, he finally showed mild symptoms and requested medical examination.
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The Bundeswehr Institute of Microbiology performed primary diagnostics of a swab taken from a skin lesion and subsequently sequenced the sample.
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Here, we announce the available data to the scientific community.
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DNA extraction was performed using DNeasy Mini Kit (Qiagen, Hilden, Germany) from clinical material and eluted in 100µl EB-Buffer.
- DNA concentrations were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Dreieich, Germany) according to the manufacturers’ protocols.
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From total DNA, an Illumina-compatible library was prepared (NEBNext® Ultra™ II DNA Library Prep Kit, NEB (New England Bialabs), Frankfurt am Main, Germany) and sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) using 2x 150bp v2 chemistry in order to obtained paired-end reads.
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Raw reads were assigned by Kraken 2 [1] and human reads were discarded.
- Remaining paired-end reads were assembled de-novo using an in-house script based on the SPAdes assembler [2] in single-cell mode.
- In addition, viral reads were mapped to MPXV_USA_2022_MA001 (AccNo:ON563414 77) for validation and manual curation of the reported genome sequence.
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Afterwards, a contig extension was performed using the previously assembled contigs.
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The full-length genome comprises 197.378 bp and was sequenced directly from clinical material.
- BLAST analysis [3] and phylogenetic inference support the classification of this isolate into the West-African clade associated with the recent isolates from Europe and the US.
- The nearest neighbor is PT0010/2022, an isolate from Portugal (published May 23 by INSA).
- All of the identified SNPs compared to the MPXV_UK_P2 34 are either TC→TT or GA→AA in dinucleotide context and potentially caused by APOBEC3 as hypothesized by Rambaut.
- In addition, a 10 bp deletion (CAATCTTTCT) was discovered at 133.175 bp which is part of an exact tandem repeat or an inexact triplet repeat upstream of a hypothetical protein.
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Interestingly, this duplication is not annotated in the recently published CDC strain ON563414 77 and the Belgium strain ITM_MPX_1_Belgium but in all sequences from Portugal (PT0001-PT0009 4).
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The genome sequence was submitted to NCBI Nucleotide (ON568298 159) and will be updated (if needed) as soon as data from the grown cell-cultures are available.
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Phylogenetic analysis is based on an incremental analysis of all publicly available full-length (>180 kbp) sequences acquired via NCBI Nucleotide or shared here by colleagues on virological.org 19.
- Recombinant or apparently partially older sequences were discarded.
- Multiple sequence alignments were carried out using MAFFT v7.490 (options: --auto --6merpair; [4]).
- Phylogenetic inference with maximum likelihood as implemented in fasttree [5] using the GTRCAT model with subsequent rescaling of branch lengths optimizing a discrete gamma model with 20 rate categories (fasttreemp option: -nt -gtr --gamma; binary compiled with -DUSE_DOUBLE).
- The full tree was rooted at the separation between the Central and West African clades and sequences of the West African Clade were selected for a reanalysis that included also the most recent sequences from Portugal 4.
- It was rooted at the midpoint and is shown below.