Bert Vanmechelen, Tony Wawina-Bokalanga and Piet Maes.
A Monkeypox virus genome was obtained from a second Belgian case at the Rega Institute, KU Leuven. Viral DNA was extracted from each swab sample (three in total) using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions. Subsequently, a conventional PCR was performed with Monkeypox-specific primers, confirming the presence of Monkeypox virus DNA in each extract.
Sequencing libraries were prepared using the ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies (ONT). We started with 400 ng of each DNA extract in 25 μL, which was end-prepped using the NEBNext Ultra II End Repair/dA-tailing module kit. The end-prepped products were purified using a 1x AMPure bead clean-up with two 80% EtOH washes. AMX adapter was ligated to the end-prepped DNA as per the SQK-LSK109 kit protocol, followed by a 0.4x AMPure bead clean-up. During this clean-up, two of the DNA samples were washed with Long Fragment buffer (LFB), while one sample was washed with Short Fragment Buffer (SFB). The final libraries were eluted in 15 μL Elution Buffer (EB) and 70-120 ng of each library was loaded onto an R9.4.1 flow cell, which were run for 72 hours.
Data from all three runs was mapped to Monkeypox virus strain Israel_2018 (GB: MN648051.1) using CLC Genomics Workbench v22.0.1. From this read mapping, a consensus sequence with an average coverage of 116x (range 24x to 192x) was extracted, which was polished using Medaka v1.4.3. Remaining errors were corrected by visual inspection of a mapping of all reads against the Medaka-polished consensus using CLC Genomics Workbench v22.0.1.
mpx_UZ-patient_1_CONSENSUS-v8.fasta.gz (56.2 KB)
Raw data available here: https://zidu.be/mpx_UZ-patient_1_rawdata.fastq.gz