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) 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 3), thus enabling future
identification of novel variants.
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 ) using bwa (v 2.2.1 ). 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|