Sandra Martínez-Puchol 1,2,3 , Andreu Coello 2,3 , Antoni E. Bordoy 2,3 , Laia Soler 3 , David Panisello 2 , Sara González-Gómez 2 , Gemma Clarà 3 , Alexia Paris de León 3 , Anna Not 2 , Águeda Hernández 3 , Silvia Bofill-Mas 4,5 , Verónica Saludes 2,3,6,* , Elisa Martró 2-4,6,* , Pere Joan Cardona 2,3,7,*
... As of 27th May 2022, eighty-four confirmed MPXV cases have been reported in Spain (https://monkeypox.healthmap.org/ 5). The Spanish case reported here corresponds to a young individual fulfilling clinical and epidemiological criteria. Whole-genome sequencing and phylogenetic analysis were performed. A first draft genome with 98.6% coverage and an average depth of 77x was reconstructed. Preliminary phylogenetic analysis of this genome together with MPVX genomes publicly available from Portugal, Belgium, France, Germany and the Netherlands showed evidence of a close genetic relationship within the West African clade. Specifically, ≤4 SNPs were identified versus Portugal sequences, and two SNPs versus the German sequence (Fig. 1).
Methods
Briefly, the clinical sample (skin swab in 400 l of viral transport media) was frozen and thawed three times to disrupt cell membranes. After a centrifugation step at 3000 rpm for 5 min, supernatant was treated with DNAse and RNAse [1]. Nucleic acid extraction was performed using QiAmp Viral RNA Mini Kit (Qiagen®) from DNase/RNase-treated clinical material, eluting in 80 μL AVE-buffer. Sequence-independent single primer amplification (SISPA) was performed on 32 μL of the extracted DNA [1]. Then, the DNA sequencing library was prepared from the SISPA purified product (85.6 ng of DNA) using the Ligation Sequencing Kit (SQK-LSK109, NEB®) with a final volume of 15 μL and a DNA quantity of 14.5 ng. The library was loaded onto an R9.4.1 flow cell which run for 19 hours on a MinION Mk1C device. ...
