Guillaume Croville 1, Mathilda Walch1, Jean-Luc Guérin1, Jean-Michel Mansuy2, Christophe Pasquier2, Jacques Izopet2
1IHAP, Université de Toulouse, INRAE, ENVT, 31300 Toulouse, France.
2CHU Toulouse, Hôpital Purpan, Virology Laboratory, 31300 France.
contact: [email protected]
A full Monkeypox virus genome was obtained from a male patient sampled at the University Hospital Center of Toulouse Purpan (France) in May 2022. We applied an Oxford Nanopore Technologies sequencing method directly on the clinical material as already done before by our team for the sequencing of an avipoxvirus a.
Viral DNA was extracted from a swab sample using the QIAamp DNA blood kit (QIAGEN) according to the manufacturer’s recommendations. One microliter of the DNA was analyzed using the Genomic DNA kit (Agilent) on the 4200 TapeStation System (Agilent). The DNA concentration of the sample was 16 ng/µL and the peaks profile ranged between 164 and 1059 bp, these results allowed a rough estimate of an average fragment size of 700 bp.
The DNA library was prepared from 320 ng of DNA using the SQK-LSK110 kit (Oxford Nanopore Technologies) and the NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England BioLabs). The preparation was performed following the ONT ligation sequencing gDNA protocol. The final library was eluted in 15 μL Elution Buffer (EB) and 44 fmol (corresponding to 20 ng of 700 bp DNA fragments) of the library was loaded onto an R9.4.1 flow cell which was run for 24 hours on a MK1C device (Oxford Nanopore Technologies). The MK1C basecalling mode was set as fast and the Qscore filtering value was set to 8. The bioinformatics protocol and software informations are detailed below.
The sequencing run produced 5 Gb of data and a total of 12 918 reads out of 7,9M (0,16%) were mapped to a closely related reference genome (MT903343), with a mean sequencing depth of 38x.
A consensus sequence of 196 923 bp MPXV_FRA_2022_TLS67 was generated using iVar software and released on GenBank: ON602722.
Bioinformatics protocol:
minimap2 -ax map-ont reference.fa mysample.fastq > mysample.sam
samtools sort mysample.sam > mysample.sort.bam
samtools index mysample.sort.bam
samtools flagstat mysample.sort.bam > mysample_flagstat
samtools depth 67.sort.bam > mysample_samdepth
samtools mpileup -A -Q 0 mysample.sort.bam | ivar consensus -p prefix -q 10 -t 0 -m 1
Softwares
minimap2 v2.5 b
samtools v1.14 c
iVar 1.3 d
References
a Croville G, Le Loc’h G, Zanchetta C, Manno M, Camus-Bouclainville C, Klopp C, Delverdier M, Lucas MN, Donnadieu C, Delpont M, Guérin JL. Rapid whole-genome based typing and surveillance of avipoxviruses using nanopore sequencing. J Virol Methods. 2018 Nov;261:34-39. https://doi.org/10.1016/j.jviromet.2018.08.003/
b Heng Li, Minimap2: pairwise alignment for nucleotide sequences, Bioinformatics , Volume 34, Issue 18, 15 September 2018, Pages 3094–3100, Minimap2: pairwise alignment for nucleotide sequences | Bioinformatics | Oxford Academic
c Petr Danecek, James K Bonfield, Jennifer Liddle, John Marshall, Valeriu Ohan, Martin O Pollard, Andrew Whitwham, Thomas Keane, Shane A McCarthy, Robert M Davies, Heng Li, Twelve years of SAMtools and BCFtools, GigaScience , Volume 10, Issue 2, February 2021, giab008, https://doi.org/10.1093/gigascience/giab008
d Grubaugh, N.D., Gangavarapu, K., Quick, J. et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol 20, 8 (2019). An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar | Genome Biology | Full Text