Whole genome sequences of Monkey pox virus from two cases in Madrid, Spain, May-June 2022
Borja de la Hoz-Sánchez, Marcos López-Ortiz, Almudena Gutiérrez-Arroyo, Patricia Roces-Álvarez, Fernando Lázaro-Perona, Elias Dahdouh, Iván Bloise, Julio García-Rodríguez, Jesús Mingorance
Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Madrid, Spain
The rapid and broad spreading of the current monkeypox outbreak  underlines the need of tools for rapid surveillance and monitoring of the virus’s sequence evolution. Several reports of draft genome sequences obtained directly from clinical samples or from viral cultures have been published in virological.org. Here we present two genome sequences assembled by mapping long and short reads obtained by direct sequencing from vesicle fluid.
The first sample (4061) was collected on May 20th, 2022 from vesicles in the pubic region of a male patient, and the second (8887) was collected on June 6th, 2022 from rectal vesicles of another male patient. Both were taken at Hospital La Paz in Madrid, Spain. None of the patients reported travelling or links to other known cases.
DNA was extracted from the samples using Qiagen’s DNeasy Blood and Tissue kit. Long read sequences were obtained with a MinionTM Mk1C sequencer (Oxford Nanopore Technology®) using the rapid library preparation kit SQK-RAD004 and a Flongle flowcell through a 24-hour run. For short read sequencing, DNA was sheared with a Covaris M220 ultrasonicator and libraries constructed with the NEBNext® Fast DNA Library Prep Set for Ion Torrent™. Fragment sizes around 350 bp were selected with an E-Gel™ Power Snap Electrophoresis System and sequenced in an Ion Torrent S5TM system.
The sequence of Monkeypox virus isolate MPXV_USA_2022_MA001 ([ON563414]) was used as the reference sequence. Long and short reads were mapped against this reference sequence using Minimap2  in Geneious Prime® 2022.0.1. Sample 4061 yielded 123,183 long reads and 1,388,065 short reads of which 2,411 (1.9%) and 35,644 (2.5%) mapped to the viral genome, respectively. Sample 8887 yielded 183,085 long reads and 2,238,210 short reads of which 1,435 (0.7%) and 40,527 (1.8%) mapped to the viral genome, respectively. The aligned reads have been deposited in Genbank under Bioproject PRJNA851991. In both cases the mapping spanned the whole reference sequence with no gaps and had mean coverages of 77x and 71x. Using only short reads left some small gaps, mostly in the ITR repeat regions. Consensus sequences were aligned to the reference sequence with MAFFT. There were few discrepancies and in most cases these were in the number of A’s or T’s in homopolymeric runs that were manually corrected after visual inspection of the mapped reads. These samples had Ct values close to 20 in a commercial real time PCR assay (Roche) and in the assay described by Li et al. . The same protocols yielded poor coverage in two samples with Ct values >25.
The consensus sequence obtained from sample 4061 (ON838940) was 100% identical to the reference sequence. The sequence from sample 8887 (ON838939) had two single nucleotide polymorphisms (SNPs): G90835A that would lead to a change from aspartic acid to asparagine at position 192 in gp096 (coding for a DNA topoisomerase), and G94325A, a silent mutation in the virion core protein gene gp099. Comparison with the sequences in the GISAID database showed that these two SNPs were unique to this sequence. For samples with high viral loads (Ct values <25) the combination of long and short reads obtained by sequencing total DNA from vesicle fluid may generate good quality assemblies useful for outbreak monitoring.
 World Health Organization (17 June 2022). Disease Outbreak News; Multi-country monkeypox outbreak in non-endemic countries: Update. Available n.d. Multi-country monkeypox outbreak: situation update.
 Li H. Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics 2018;34:3094–100. Minimap2: pairwise alignment for nucleotide sequences | Bioinformatics | Oxford Academic.
 Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA. J Virol Methods 2010;169:223–7. Redirecting.