MinIon-produced Illumina-confirmed genomes of the two first isolated cases from France

MinIon-produced Illumina-confirmed genomes of the two first isolated cases from France.

Gorgé, O., Jarjaval, F, Nolent, F., Criqui, A., Lourenco, J., Badaoui, A., Khoury, R., Burrel, S. Lamer, O., Chapus, C., Simon-Loriere, E., Tournier, J.N., Ferraris, O.

Monkeypox virus genomes were isolated from the first two French cases received at the National Reference Center for orthopoxviruses (CNR OPXV). The two isolates, from male patients, were collected at the CNR OPXV on May 19th and 20th. The first patient (IRBA22-11) declared no history of overseas traveling, while the second (IRBA22-14) participated to an international gathering at the Canary Islands, Spain.

Viral DNA was extracted from each sample using the Macherey Nagel pathogen kit according to the manufacturer’s instructions in two rounds. The first one was used for RT-q-PCR. It was performed with Monkeypox-specific primers and probe developed by the lab in the frame of its mandate of CNR OPXV. This assay confirmed the presence of Monkeypox virus DNA in each extract on the 20th. The clade confirmation was done also with specific RT-q-PCR developed by the CNR OPXV. The second round was dedicated to sequencing and included a DNAse treatment before extraction. The aim was to remove free host DNA and thus to enrich the extract on viral DNA.

Sequencing libraries were prepared in parallel for Oxford Nanopore Technologies (ONT) and Illumina. For ONT, we used the ligation sequencing kit (SQK-LSK109) after whole genome amplification (Cytiva kit, FisherScientific, Illkirch, France), following the manufacturer’s instructions. We started with 200 µL of each extract, and prepared four libraries per extract. During final clean-up, two libraries per patient were washed with Long Fragment buffer (LFB) and two with Short Fragment Buffer (SFB). The final libraries were eluted in 13 μL Elution Buffer (EB). Two R9.4.1 flow cells were loaded each with four libraries (200 ng per library) and were run for 10 hours.

Data from all runs were filtered with kraken/bracken (v2.1.2/v2.6.2) to get reads identified as chordopoxvirinae family. We merged reads obtained with the two washing buffers and then mapped to Monkeypox virus strain Israel_2018 (MN648051.1) with minimap2 v2.24. From this reads mapping, a consensus sequence was computed for each sequence by using nanopolish (v 0.13.2) and bcftools (v1.14). We ended up with 53632 mapped reads out of 130176 raw reads and a depth of 620x for IRBA22-11, and 98283 mapped reads out of of 348523 raw reads and a depth of 1368x for IRBA22-14.

In parallel, libraries were prepared for Illumina with the NEBNext UltraII DNA kit (New England Biolabs, Ipswich, MA, USA), after shearing with M220 Covaris device (Covaris, Woburn, MA, USA), accordingly with manufacturer’s recommendations. Non amplified (“Native”) as well as amplified extracts were processed, owing the fact that NEBNext UltraII kit needs much less input DNA than ONT. After size selection with AMPure beads (Beckman, Brea, CA, USA), sequencing was achieved using a HT 2x150bp cartridge on NextSeq 550 instrument (Illumina, San Diego, CA, USA).

We mapped the reads with bwa mem (v0.7.17-r1188) and obtained four datasets with a tremendous difference between native and amplified samples. We get respectively 46.2 and 66.3% of mapped reads for IRBA22-11 and IRBA22-14 after amplification, and only 9.5 and 6.9% for native extracts. We attributed the difference to the additional DNAse treatment done for amplified samples. There was no nucleotide difference among the two sequences obtained from each sample, indicating that amplification did not alter original sequences.

We then aggregate the sequencing data from the two platforms. We tried to use Unicycler (v0.4.8) for merging the data but unsuccessfully. We assume that reads from ONT were not long enough to fit with software requirements. Thus, we used the Nanopore consensus obtained from minimap2 as references for mapping the Illumina reads. We filtered the sam file to remove unmapped reads and used Geneious R11 (Biomatters, Auckland, New Zealand) to compare mapped reads to consensus sequences. We corrected only one base, seven T being called by ONT instead of the 6 T stretch present in locations 15951-15956.

We align these two new French sequences to the released ones by using Nextstrain tool. The two sequences are well located within the 2022 outbreak phylogenetic cluster, confirming that the cases are molecularly related. Interestingly, the one originating from the first patient (IRBA22-11), but with no direct link with presumed initial site of contamination for Europe, exhibited one SNP compared to the most frequent sequence in Europe (C148482T), possibly indicating a micro-evolution.

The two consensus sequences have been deposited on GenBank (ON755039 for IRBA22-11 and ON755040 for IRBA22-14).

Acknowledgements: this work was done in the frame of the mission of French National Reference Center for Orthopoxviruses, with the support of Santé Publique France.