# Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen

Aurelia Kwasiborski, Véronique Hourdel, Charlotte Balière, Damien Hoinard, Quentin Grassin, Maxence Feher, Clémentine de la Porte Des Vaux, Mélanie Cresta, Jessica Vanhomwegen, Jean-Claude Manuguerra, et al.

► To cite this version:

Aurelia Kwasiborski, Véronique Hourdel, Charlotte Balière, Damien Hoinard, Quentin Grassin, et al. Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen. Microbiology Resource Announcements, 2024, 13 (1), pp.e0081123. 10.1128/MRA.00811-23. pasteur-04424081

HAL Id: pasteur-04424081

https://pasteur.hal.science/pasteur-04424081

Submitted on 29 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen

Aurelia Kwasiborski,¹ Véronique Hourdel,¹ Charlotte Balière,¹ Damien Hoinard,¹ Quentin Grassin,¹ Maxence Feher,¹ Clémentine De La Porte Des Vaux,² Mélanie Cresta,² Jessica Vanhomwegen,¹ Jean-Claude Manuguerra,¹ Christophe Batéjat,¹ Valérie Caro¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 2.

**ABSTRACT** We report the whole-genome sequence of monkeypox virus obtained using MinION technology (Oxford Nanopore Technologies) from a French clinical specimen during the 2022 epidemic. Amplicon-based sequencing and shotgun metagenomic approaches were directly applied to the sample.

**KEYWORDS** monkeypox virus, metagenomics, amplicon-based sequencing, nanopore, next-generation sequencing

Monkeypox virus (MPXV), a double-stranded DNA virus with a genome of approximately 200 kb and belonging to the genus *Orthopoxvirus* of the family *Poxviridae*, is the causative agent of monkeypox recently renamed mpox (1). In 2022, this zoonotic disease was responsible for the most widespread and largest outbreak outside Africa (2). This global outbreak has strengthened the need for rapid genomic-based surveillance tools. Here, we report the genomic sequence of MPXV based on a comparison between direct metagenomic sequencing as already referenced (3, 4) and an amplicon-based approach using the MinION.

An oropharyngeal swab was collected from a 35-year-old French man who consulted Bichat-Claude Bernard Hospital, Paris France, in June 2022, for mpox suggestive lesions and was sent to the Laboratory for Urgent Response to Biological Threats for diagnostic testing (specimen identification number CIBU-220031).

Genomic DNA was extracted using the QIAamp DNA blood kit (QIAGEN) according to the manufacturer's recommendations. The clinical specimen was found positive with a threshold cycle value of 23 using an MPXV real-time PCR assay targeting the *G2RG* gene (5).

For the metagenomics approach (CIBU-220031-M), a DNA library was prepared from 1 ng of DNA using the Rapid PCR Barcoding kit (SQK-RPB004) and loaded onto an R9.4.1 flow cell for 24 hours of sequencing run on the Mk1C device. The nextflow workflow (wf-mpx version 0.0.4) was applied to FastQ files with medaka (1.5.0) for mapping to the reference MT903344, flye (2.9-b1768) for assembly and medaka (1.5.0), bedtools (v2.30.0), and bcftools (1.15) for consensus generation (6, 7).

For the amplicon-based method (CIBU-220031-A), a multiplex MPXV PCR protocol was applied to genomic DNA (8). Obtained PCR products were purified using a 1:1 ratio of Ampure XP clean up beads (Beckman Coulter). The library was prepared using Ligation Sequencing (SQK-LSK109) and Native Barcoding kits (EXP-NBD104/114) and loaded onto R9.4.1 flow-cell for 24 hours on the Mk1C. The field bioinformatics protocol proposed by the ARTIC Network was adapted for data analysis and consensus sequence generation. Guppy (v6.0.7), minimap2 (v2.17-r941) (9), and medaka (v1.0.3) were, respectively, used for baseCALLing, de-multiplexing, mapping, and consensus generation. Cramino (v0.9.7) and Tablet (v1.21.02.08) (10) were used to calculate the number of reads and the percentage of genome coverage and depth. Default parameters were applied.

**Editor** Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, Belgium

Address correspondence to Valérie Caro, valerie.caro@pasteur.fr.

The authors declare no conflict of interest.

See the funding table on p. 2.

**Received** 30 August 2023

**Accepted** 30 October 2023

**Published** 4 December 2023

Copyright © 2023 Kwasiborski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
# Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen

Aurelia Kwasiborski,¹ Véronique Hourdel,¹ Charlotte Balière,¹ Damien Hoinard,¹ Quentin Grassin,¹ Maxence Feher,¹ Clémentine De La Porte Des Vaux,² Mélanie Cresta,² Jessica Vanhomwegen,¹ Jean-Claude Manuguerra,¹ Christophe Batéjat,¹ Valérie Caro¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 2.

**ABSTRACT** We report the whole-genome sequence of monkeypox virus obtained using MinION technology (Oxford Nanopore Technologies) from a French clinical specimen during the 2022 epidemic. Amplicon-based sequencing and shotgun metagenomic approaches were directly applied to the sample.

**KEYWORDS** monkeypox virus, metagenomics, amplicon-based sequencing, nanopore, next-generation sequencing

Monkeypox virus (MPXV), a double-stranded DNA virus with a genome of approximately 200 kb and belonging to the genus *Orthopoxvirus* of the family *Poxviridae*, is the causative agent of monkeypox recently renamed mpox (1). In 2022, this zoonotic disease was responsible for the most widespread and largest outbreak outside Africa (2). This global outbreak has strengthened the need for rapid genomic-based surveillance tools. Here, we report the genomic sequence of MPXV based on a comparison between direct metagenomic sequencing as already referenced (3, 4) and an amplicon-based approach using the MinION.

An oropharyngeal swab was collected from a 35-year-old French man who consulted Bichat-Claude Bernard Hospital, Paris France, in June 2022, for mpox suggestive lesions and was sent to the Laboratory for Urgent Response to Biological Threats for diagnostic testing (specimen identification number CIBU-220031).

Genomic DNA was extracted using the QIAamp DNA blood kit (QIAGEN) according to the manufacturer's recommendations. The clinical specimen was found positive with a threshold cycle value of 23 using an MPXV real-time PCR assay targeting the *G2RG* gene (5).

For the metagenomics approach (CIBU-220031-M), a DNA library was prepared from 1 ng of DNA using the Rapid PCR Barcoding kit (SQK-RPB004) and loaded onto an R9.4.1 flow cell for 24 hours of sequencing run on the Mk1C device. The nextflow workflow (wf-mpx version 0.0.4) was applied to FastQ files with medaka (1.5.0) for mapping to the reference MT903344, flye (2.9-b1768) for assembly and medaka (1.5.0), bedtools (v2.30.0), and bcftools (1.15) for consensus generation (6, 7).

For the amplicon-based method (CIBU-220031-A), a multiplex MPXV PCR protocol was applied to genomic DNA (8). Obtained PCR products were purified using a 1:1 ratio of Ampure XP clean up beads (Beckman Coulter). The library was prepared using Ligation Sequencing (SQK-LSK109) and Native Barcoding kits (EXP-NBD104/114) and loaded onto R9.4.1 flow-cell for 24 hours on the Mk1C. The field bioinformatics protocol proposed by the ARTIC Network was adapted for data analysis and consensus sequence generation. Guppy (v6.0.7), minimap2 (v2.17-r941) (9), and medaka (v1.0.3) were, respectively, used for baseCALLing, de-multiplexing, mapping, and consensus generation. Cramino (v0.9.7) and Tablet (v1.21.02.08) (10) were used to calculate the number of reads and the percentage of genome coverage and depth. Default parameters were applied.

**Editor** Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, Belgium

Address correspondence to Valérie Caro, valerie.caro@pasteur.fr.

The authors declare no conflict of interest.

See the funding table on p. 2.

**Received** 30 August 2023

**Accepted** 30 October 2023

**Published** 4 December 2023

Copyright © 2023 Kwasiborski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<table><caption>TABLE 1 Nanopore sequencing data for sample CIBU-220031-M and CIBU-220031-A</caption><thead><tr><td>Feature</td><td>CIBU-220031-M</td><td>CIBU-220031-A</td></tr></thead><tbody><tr><td>No. of total reads (M)</td><td>4.27</td><td>3.33</td></tr><tr><td>No. of mapped reads (M)</td><td>34,760</td><td>59,794</td></tr><tr><td>Mean length of reads (bp)</td><td>2,868</td><td>2,073</td></tr><tr><td>Mean quality score</td><td>11.6</td><td>12</td></tr><tr><td>Average depth (x)</td><td>462</td><td>577</td></tr><tr><td>Maximum coverage depth</td><td>733</td><td>800</td></tr><tr><td>Consensus length (bp)</td><td>197,233</td><td>197,179</td></tr><tr><td>GC content (%)</td><td>33</td><td>33</td></tr></tbody></table>

The runs respectively generated 4.27 and 3.33 million reads for CIBU-220031-M and CIBU-220031-A (Table 1). Mapping against the reference MT903344 showed 99.97% and 99.42% genome coverage with an average depth of 462x and 577x, respectively. Consensus sequences of 197,233 bp for CIBU-220031-M and 197,179 bp for CIBU-220031-A were generated. The length difference is due to the failure to obtain the genomic ends with the amplicon-based method. Both viral genome sequences showed 100% nucleotide identity between them and 99.2% nucleotide identity with the reference MT903344, unveiling 47 nucleotide substitutions (5 out of CDS, 19 synonymous, and 23 non-synonymous). The viral sequence was lineage B.1. in clade IIb according to the Nextclade application (11).

Both approaches have proved successful in obtaining correct genomic sequences. However, the amplicon-based approach yields more targeted reads, increasing the depth coverage. Thus, this method would enable more samples to be sequenced in a single MinION run, reducing the cost and time, which can be important factors when dealing with an infectious disease.

## ACKNOWLEDGMENTS

This project received funding from the French Institut Carnot "Microbes-Santé" in collaboration with the "France Futur Elevage" Carnot of INRAE Institute, in the framework of the FIELD project.

## AUTHOR AFFILIATIONS

## AUTHOR ORCIDs

Valérie Caro http://orcid.org/0000-0001-8477-5708

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Association Instituts Carnot (Instituts Carnot)</td><td>FIELD</td><td>Aurelia Kwasiborski<br>Véronique Hourdel<br>Charlotte Balière<br>Damien Hoinard<br>Quentin Grassin<br>Maxence Feher<br>Jessica Vanhomwegen<br>Jean-Claude Manuguerra<br>Christophe Batéjat</td></tr></tbody></table>

<footnote n="1">¹Institut Pasteur, Université Paris Cité, Environment and Infectious Risks Unit, Laboratory for Urgent Response to Biological Threats, Paris, France</footnote>

<footnote n="2">²Bichat-Claude Bernard Hospital, Paris, France</footnote>
<table><caption>TABLE 1 Nanopore sequencing data for sample CIBU-220031-M and CIBU-220031-A</caption><thead><tr><td>Feature</td><td>CIBU-220031-M</td><td>CIBU-220031-A</td></tr></thead><tbody><tr><td>No. of total reads (M)</td><td>4.27</td><td>3.33</td></tr><tr><td>No. of mapped reads (M)</td><td>34,760</td><td>59,794</td></tr><tr><td>Mean length of reads (bp)</td><td>2,868</td><td>2,073</td></tr><tr><td>Mean quality score</td><td>11.6</td><td>12</td></tr><tr><td>Average depth (x)</td><td>462</td><td>577</td></tr><tr><td>Maximum coverage depth</td><td>733</td><td>800</td></tr><tr><td>Consensus length (bp)</td><td>197,233</td><td>197,179</td></tr><tr><td>GC content (%)</td><td>33</td><td>33</td></tr></tbody></table>

The runs respectively generated 4.27 and 3.33 million reads for CIBU-220031-M and CIBU-220031-A (Table 1). Mapping against the reference MT903344 showed 99.97% and 99.42% genome coverage with an average depth of 462x and 577x, respectively. Consensus sequences of 197,233 bp for CIBU-220031-M and 197,179 bp for CIBU-220031-A were generated. The length difference is due to the failure to obtain the genomic ends with the amplicon-based method. Both viral genome sequences showed 100% nucleotide identity between them and 99.2% nucleotide identity with the reference MT903344, unveiling 47 nucleotide substitutions (5 out of CDS, 19 synonymous, and 23 non-synonymous). The viral sequence was lineage B.1. in clade IIb according to the Nextclade application (11).

Both approaches have proved successful in obtaining correct genomic sequences. However, the amplicon-based approach yields more targeted reads, increasing the depth coverage. Thus, this method would enable more samples to be sequenced in a single MinION run, reducing the cost and time, which can be important factors when dealing with an infectious disease.

## ACKNOWLEDGMENTS

This project received funding from the French Institut Carnot "Microbes-Santé" in collaboration with the "France Futur Elevage" Carnot of INRAE Institute, in the framework of the FIELD project.

## AUTHOR AFFILIATIONS

## AUTHOR ORCIDs

Valérie Caro http://orcid.org/0000-0001-8477-5708

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Association Instituts Carnot (Instituts Carnot)</td><td>FIELD</td><td>Aurelia Kwasiborski<br>Véronique Hourdel<br>Charlotte Balière<br>Damien Hoinard<br>Quentin Grassin<br>Maxence Feher<br>Jessica Vanhomwegen<br>Jean-Claude Manuguerra<br>Christophe Batéjat</td></tr></tbody></table>

<footnote n="1">¹Institut Pasteur, Université Paris Cité, Environment and Infectious Risks Unit, Laboratory for Urgent Response to Biological Threats, Paris, France</footnote>

<footnote n="2">²Bichat-Claude Bernard Hospital, Paris, France</footnote>
<table><thead><tr><td>Funder</td><td>Grant(s) Author(s)</td></tr></thead><tbody><tr><td></td><td>Valérie Caro</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Véronique Hourdel, Data curation, Formal analysis, Methodology, Software, Writing – original draft | Charlotte Balière, Formal analysis, Methodology, Writing – review and editing | Damien Hoinard, Formal analysis, Methodology | Quentin Grassin, Formal analysis, Methodology | Maxence Feher, Formal analysis, Methodology | Clémentine De La Porte Des Vaux, Investigation | Mélanie Cresta, Investigation | Jessica Vanhomwegen, Funding acquisition, Methodology, Writing – review and editing | Jean-Claude Manuguerra, Supervision, Writing – review and editing | Christophe Batéjat, Data curation, Funding acquisition, Supervision | Valérie Caro, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing.

## DATA AVAILABILITY

The consensus genome sequence was deposited in the GISAID EpiPox database (accession number EPI_ISL_18128768) and GenBank (accession number OR473631). The raw reads were deposited in the NCBI Sequence Read Archive under accession number PRJNA1004259.

## ETHICS APPROVAL

The lab investigations described in this article were carried out in accordance with the General Data Protection Regulation [Regulation (EU) 2016/679 and Directive 95/46/EC] and the French data protection law (Law 78-17 on 06 January 1978 and Decree 2019-536 on 29 May 2019) which does not require a review by an ethics committee for the secondary use of samples collected for healthcare purposes. In such case, the secondary use for research is authorized if the persons have been informed of such secondary use (article L.1211-2 of the French Public Health Code).

## REFERENCES

1. Isidro J, Borges V, Pinto M, Sobral D, Santos JD, Nunes A, Mixão V, Ferreira R, Santos D, Duarte S, Vieira L, Borrego MJ, Núncio S, de Carvalho IL, Pelerito A, Cordeiro R, Gomes JP. 2022. Addendum: phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med 28:2220-2221. https://doi.org/10.1038/s41591-022-02036-2

2. Mpox (Monkeypox) outbreak. 2022. Available from: https://www.who.int/emergencies/situations/monkeypox-oubreak-2022

3. Balière C, Hourdel V, Kwasiborski A, Grassin Q, Feher M, Hoinard D, Vanhomwegen J, Taieb F, Consigny PH, Manuguerra JC, Leclercq I, Batéjat C, Caro V. 2023. Complete genome sequences of monkeypox virus from a french clinical sample and the corresponding isolated strain, obtained using nanopore sequencing. Microbiol Resour Announc 12:e0000923. https://doi.org/10.1128/mra.00009-23

4. Vandenbogaert M, Kwasiborski A, Gonofio E, Descorps-Declère S, Selekon B, Nkili Meyong AA, Ouilibona RS, Gessain A, Manuguerra JC, Caro V, Nakoune E, Berthet N. 2022. Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the central African republic. Sci Rep 12:10768. https://doi.org/10.1038/s41598-022-15073-1

5. Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. 2010. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo

Basin strain DNA. J Virol Methods 169:223-227. https://doi.org/10.1016/j.jviromet.2010.07.012

6. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, Garcia MU, Di Tommaso P, Nahnsen S. 2020. The Nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol 38:276-278. https://doi.org/10.1038/s41587-020-0439-x

7. Wf-Mpx | Mpox virus Metagenomic assembly. 2023. Groovy. EPI2ME Labs.

8. Chen NFG, Gagne L, Doucette M, Smole S, Buzby E, Hall J, Ash S, Harrington R, Cofsky S, Clancy S, Kapsak CJ, Sevinsky J, Libuit K, Mallery B, Chaguza C, Grubaugh ND, Park DJ, Gallagher GR, Vogels CBF. 2022. Monkeypox Virus Multiplexed PCR Amplicon Sequencing (PrimalSeq) V.2

9. Li H. 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094-3100. https://doi.org/10.1093/bioinformatics/bty191

10. Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, Shaw PD, Marshall D. 2013. Using tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193-202. https://doi.org/10.1093/bib/bbs012

11. Aksamentov I, Roemer C, Hodcroft EB, Neher RA. 2021. Nextclade: clade assignment, mutation calling and quality control for viral genomes. JOSS 6:3773. https://doi.org/10.21105/joss.03773
<table><thead><tr><td>Funder</td><td>Grant(s) Author(s)</td></tr></thead><tbody><tr><td></td><td>Valérie Caro</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Véronique Hourdel, Data curation, Formal analysis, Methodology, Software, Writing – original draft | Charlotte Balière, Formal analysis, Methodology, Writing – review and editing | Damien Hoinard, Formal analysis, Methodology | Quentin Grassin, Formal analysis, Methodology | Maxence Feher, Formal analysis, Methodology | Clémentine De La Porte Des Vaux, Investigation | Mélanie Cresta, Investigation | Jessica Vanhomwegen, Funding acquisition, Methodology, Writing – review and editing | Jean-Claude Manuguerra, Supervision, Writing – review and editing | Christophe Batéjat, Data curation, Funding acquisition, Supervision | Valérie Caro, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing.

## DATA AVAILABILITY

The consensus genome sequence was deposited in the GISAID EpiPox database (accession number EPI_ISL_18128768) and GenBank (accession number OR473631). The raw reads were deposited in the NCBI Sequence Read Archive under accession number PRJNA1004259.

## ETHICS APPROVAL

The lab investigations described in this article were carried out in accordance with the General Data Protection Regulation [Regulation (EU) 2016/679 and Directive 95/46/EC] and the French data protection law (Law 78-17 on 06 January 1978 and Decree 2019-536 on 29 May 2019) which does not require a review by an ethics committee for the secondary use of samples collected for healthcare purposes. In such case, the secondary use for research is authorized if the persons have been informed of such secondary use (article L.1211-2 of the French Public Health Code).

## REFERENCES

1. Isidro J, Borges V, Pinto M, Sobral D, Santos JD, Nunes A, Mixão V, Ferreira R, Santos D, Duarte S, Vieira L, Borrego MJ, Núncio S, de Carvalho IL, Pelerito A, Cordeiro R, Gomes JP. 2022. Addendum: phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med 28:2220-2221. https://doi.org/10.1038/s41591-022-02036-2

2. Mpox (Monkeypox) outbreak. 2022. Available from: https://www.who.int/emergencies/situations/monkeypox-oubreak-2022

3. Balière C, Hourdel V, Kwasiborski A, Grassin Q, Feher M, Hoinard D, Vanhomwegen J, Taieb F, Consigny PH, Manuguerra JC, Leclercq I, Batéjat C, Caro V. 2023. Complete genome sequences of monkeypox virus from a french clinical sample and the corresponding isolated strain, obtained using nanopore sequencing. Microbiol Resour Announc 12:e0000923. https://doi.org/10.1128/mra.00009-23

4. Vandenbogaert M, Kwasiborski A, Gonofio E, Descorps-Declère S, Selekon B, Nkili Meyong AA, Ouilibona RS, Gessain A, Manuguerra JC, Caro V, Nakoune E, Berthet N. 2022. Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the central African republic. Sci Rep 12:10768. https://doi.org/10.1038/s41598-022-15073-1

5. Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. 2010. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo

Basin strain DNA. J Virol Methods 169:223-227. https://doi.org/10.1016/j.jviromet.2010.07.012

6. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, Garcia MU, Di Tommaso P, Nahnsen S. 2020. The Nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol 38:276-278. https://doi.org/10.1038/s41587-020-0439-x

7. Wf-Mpx | Mpox virus Metagenomic assembly. 2023. Groovy. EPI2ME Labs.

8. Chen NFG, Gagne L, Doucette M, Smole S, Buzby E, Hall J, Ash S, Harrington R, Cofsky S, Clancy S, Kapsak CJ, Sevinsky J, Libuit K, Mallery B, Chaguza C, Grubaugh ND, Park DJ, Gallagher GR, Vogels CBF. 2022. Monkeypox Virus Multiplexed PCR Amplicon Sequencing (PrimalSeq) V.2

9. Li H. 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094-3100. https://doi.org/10.1093/bioinformatics/bty191

10. Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, Shaw PD, Marshall D. 2013. Using tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193-202. https://doi.org/10.1093/bib/bbs012

11. Aksamentov I, Roemer C, Hodcroft EB, Neher RA. 2021. Nextclade: clade assignment, mutation calling and quality control for viral genomes. JOSS 6:3773. https://doi.org/10.21105/joss.03773