dataesr/scientific-paragraphs-categorization huggingface dataset:
1. Indeed, the heavily mutated RBD structure predicted by our in silico analysis shortly after the emergence of this variant was remarkably similar to posterior cryo-electron microscopy data (pdb 7t9l)  [39, 40]. SARS-CoV-2 spike gene and protein sequence analysis. SARS-CoV-2 spike sequences from humans, European minks and American minks were collected from Genbank and GISAID (Table  1 )  [17, 41]. Multiple alignments were conducted with MUSCLE using the SeaView package  [42]. The nucleic acid phylogenetic tree was built the using the maximum likelihood method under the HKY model with 500 bootstrap repeats. The protein tree was built with the maximum likelihood method using the Q. plant +G + F model with 500 bootstrap repeats. Tress were rooted using the SARS-CoV-2 original Wuhan HU1 strain spike gene or protein (NC_045512.
The mutations I692V and M1229Y were found in the same strain (EPI_ISL_615652) whereas the other three mutations were individual, each one being present in a different strain (EPI_ISL_641474, EPI_ISL_618946 and EPI_ISL_616971). Only one case of additional mutations was found in a SARS-CoV-2 strain isolated from minks only. It is the triple L5F-N751Y-C1250F in strain EPI_ISL_641413. The N571F mutation was found both in humans and minks whereas all the other 11 mutations were found only in SARS-CoV-2 strains isolated exclusively from humans. With the exception of the double A626S-N751Y mutation in the strain EPI_ISL_625720, all mutations were singletons in different human-isolated SARS-CoV-2 strains. The 3-D positioning of these mutations indicated that they were distributed in five different functional groups (Fig.
Discussion Although it cannot be absolutely ruled out that the outbreak of SARS-CoV-2 in certain mink farms could have been initiated with the introduction into the farm of a sick or asymptomatic carrier animal (e. g. , wild Mustelidae which sometimes enter farms to steal food)  [44, 45] , whole genome sequencing of viruses and phylogenic analysis evidenced that minks in Northern Europe were infected by staff members in charge of animal care and/or cage maintenance. Indeed, mink SARS-CoV-2 genomes available through the GISAID database indicated that they branched on human SARS-CoV-2 sequences and that mutants including the Y453F change occurred afterwards  [14]. The virus spreading and the accumulation of host-specific mutations were favored in mink farms by intensive breeding conditions with overcrowded animals caged This work demonstrates the pivotal role of the ACE2 amino acid 34 and the S protein amino acid 453 in the spike-ACE2 interaction during SARS-CoV-2 infection.
The S1-S2 cleavage spike region is playing a key role in potentiating the membrane fusion process and therefore increasing the overall kinetics of Sample names are built with the Genbank of GISAID accession number followed by a fourletter code (Mlut for Mustella lutreola, Nvis for Neovison vison and Hsap for Homo sapiens) identifying the species and a country code (DK for Denmark, NL for The Netherlands and USA for United States of America) indicating the geographical origin of the sample except for the sequences from China in which the city of origin is indicated. The tree was rooted using the spike gene sequence of the Wuhan Hu1 SARS-CoV-2 strain. The colour code corresponds to the nature of the 453 and 654 residues. Blue: Outgroup rooting the tree. Purple: Y453 + D614. Yellow: Y453 + D614G.
Funding This work was supported by the  French Government  under the « Investissements d'avenir » (Investments for the Future) programme managed by the  Agence Nationale de la Recherche (ANR, FR: National Agency for Research) , (reference: Méditerranée Infection  10-IAHU-03 ) to  Prof. Didier Raoult  and annual budget allocation from  Aix-Marseille Université and IRD  to the  MEPHI laboratory.
The genus Bordetella includes bacteria that are found in the environment and/or associated with humans and other animals. A few closely related species, including Bordetella pertussis, are human pathogens that cause diseases such as whooping cough. Here, we present a large database of Bordetella isolates and genomes and develop genotyping systems for the genus and for the B. pertussis clade. To generate the database, we merge previously existing databases from Oxford University and Institut Pasteur, import genomes from public repositories, and add 83 newly sequenced B. bronchiseptica genomes. The public database currently includes 2,582 Bordetella isolates and their provenance data, and 2,085 genomes (https://bigsdb. pasteur. fr/bordetella/). We use core-genome multilocus sequence typing (cgMLST) to develop genotyping systems for the whole genus and for B.
Some Bordetella species have been found in environmental samples, such as B. petrii or the three recently described species B. muralis, B. tumulicola and B. tumbae  [17] [18] [19]. In addition to these 16 current Bordetella species with standing in the prokaryotic taxonomy, Bordetella genogroups were identified from patients with cystic fibrosis based on single-gene nrdA sequencing  20  ; these are labeled as 'genomosp. ' in INSDC bioproject PRJNA385118 and may represent additional Bordetella species. The diversity of lifestyles and medical importance of Bordetellae raise important questions about the origins and evolution of pathogenicity in this group  4. Horizontal gene transfer (HGT) is likely to occur between Bordetella species and lineages, as already observed for the O-antigen locus in B.
bronchiseptica  21  , and the environmental species B. petrii has genomic islands with atypical G+C content  17  that were associated with the metabolic versatility of this species, whereas in contrast, gene gain is rare or absent in B. pertussis, which evolved mainly through gene loss  22. The use of a unified database of genomes from all Bordetellae species would facilitate genomic analyses underlying the phenotypic diversity within this important bacterial genus  23. Besides the large-scale evolution of Bordetellae, the population dynamics within Bp are an important topic of epidemiological surveillance, in light of vaccine-driven evolution and the possible emergence and global dissemination of antimicrobial resistance  [24] [25] [26]. Major evolutionary events in vaccine antigen-related genomic features have been described, including within pertussis toxin subunit A (ptxA), the promoter region of the pertussis toxin gene cluster (ptxP), and the fimbriae gene fim3  24.
Bioinformatics platforms that centralize allele definitions and allow curation and open access to genotype nomenclature are critical resources for unified pathogen subtype definitions  29. Until now, there have been two dedicated Bordetella genome sequence databases: Oxford University's PubMLST (unpublished) and Institut Pasteur's BIGSdb  30  platforms. This duality has led to nomenclatural confusion and complexity for users, who may need to consult two distinct databases. With the rapid developments of genomic epidemiology and biodiversity surveys, a common and optimized resource for Bordetella evolutionary studies and sequence variants naming is needed. In this work, we set-up such a unified genomic platform at https://bigsdb. pasteur. fr/bordetella/ and expand its features to enable addressing genus-wide evolutionary genomics questions and global tracking of important genotypes within individual species, and particularly within B.
Phylogenetic analysis of the genus The average nucleotide identity (ANI) metric is now universally used as a guide to define novel bacterial species when genomic sequences are available  31. ANI values between representative genomes of the Bordetella genus (Supplementary Data 1; Supplementary Fig. 1 ) showed that most currently described species differ by >5% nucleotide positions. A well-known exception is represented by the classical Bordetella members, which present ANI values between 98. 3 and 99. 4% among them, and which belong to a single genomic species, which we have labeled here the B. bronchiseptica genomic species (BbGS). We defined genomic species as groups of genomes with >95% ANI and numbered these from The genus-wide cgMLST scheme loci covered a ~20-30% fraction of the entire length of Bordetella genomes, depending on species (Supplementary Data 3).
Phylogenetic diversity within the Bordetella bronchiseptica genomic species We gathered all unique isolates identified as B. bronchiseptica and added 83 B. bronchiseptica isolates from France that were sequenced for this study into a single public project (project id 24: B. bronchiseptica phylogeny, see Supplementary Data 4). After automated filtering of genomes exhibiting more than 25% of missing data, a phylogenetic analysis was performed with representatives of B. pertussis and B. parapertussis and revealed a primary separation into two deep lineages (Figure  2  & Supplementary Fig. 3 ). We define the minor one as lineage II of B. bronchiseptica. Lineage II comprised 13 isolates, including HT200, collected from a natural spring water in India and previously recognized as being atypically distant from other Bbs isolates  33.
In our dataset, considering isolates collected after 2008, ptxP1 isolates mainly originated from China, whereas ptxP3 isolates were predominantly from France and the USA (Supplementary Fig. 6 ). The ptxP3 branch is itself subdivided by allelic variation of the fim3 gene (fim3-1 and fim3-2 genotypes). Variation observed at the ptxA1, ptxP and fim3 loci has previously been attributed to vaccine-driven evolution  24. Pertactin (PRN) is another important vaccine antigen of B. pertussis. Even though this adhesin is not expressed in a large fraction of currently circulating isolates in countries that use acellular vaccines  28, 39, 40  , its genetic variation also marks important phylogenetic subdivisions  24. We included the prn locus in our database within the Autotransporters scheme (scheme id: 12).
Macrolide resistance markers in Bordetella pertussis To detect alleles associated with macrolide resistance, Bp isolates were screened with the dedicated macrolide resistance scheme, which comprises the gene coding for 23S rRNA, and fhaB. Allele 13 of the 23S_rRNA locus, characterized by the A2047G mutation, is specific for macrolide-resistant isolates, whereas alleles 1, 2 and 20 correspond to susceptible ones. Allele 13 was found in 46 of 51 isolates from China  25  and in two isolates previously described as erythromycin resistant: one from USA (ATCC:BAA-1335D-5,  41  ) and one from France (FR4991,  42  ). When checking our entire database, which contained 1,566 Bp isolates, no additional resistant isolate was found. In a previous study, Xu et al. 25  showed that allele fhaB3 characterized by the mutation C5330T was associated with macrolide-resistant isolates from China.
BIGSdb data visualization functionality BIGSdb offers several possibilities to visualize data from the genomic database  29  including the use of built-in plugins that connect to external tools such as GrapeTree 43 or iTOL  44. We illustrate the use of GrapeTree by visualizing the diversity of cgMLST profiles of B. pertussis, B. parapertussis and B. bronchiseptica genomes (Supplementary Fig. 7 ). This analysis illustrates that the main lineages of B. bronchiseptica are distinguished using cgMLST profilebased comparisons, and how the diversity of each sublineage is internally structured into cgMLST types. In turn, the iTOL plugin can be used to build phylogenetic trees based on sequence alignments; we illustrate this with the nrdA gene, which was initially used to define genogroups  20. The nrdA-based phylogenetic tree (Supplementary Fig.
Discussion We have set-up a unified genomic platform, available at https://bigsdb. pasteur. fr/bordetella/, by merging the data from the two previously existing PubMLST and Pasteur BIGSdb-Bordetella databases. This merger has enabled allelic nomenclature unification and has led to reassemble on the same platform, genotyping schemes that will facilitate the study of Bordetella diversity at different phylogenetic breadths. We have also expanded the genomic resource with public sequences and sequenced genomes from the French NRC. The resulting unified platform for Bordetella genotyping will ease the future curation and expansion of Bordetella genomic data by a community of users and curators. Two complementary cgMLST schemes are available on the genomic platform. While the cgMLST_genus scheme should be preferred for genus-wide phylogenetic analyses and within individual species including B.
bronchiseptica, the cgMLST_pertussis scheme comprises more loci that are conserved within Bp, and its added discriminatory power will be useful for strain identification and comparison within Bp. Recently, a whole-genome MLST (wgMLST) scheme comprising even more loci (n =3,506; 1,822 of which are common with our cgMLST_pertussis scheme) was proposed for Bp subtyping, and concordant results were obtained for classification of outbreak and sporadic isolates from the USA using the cgMLST and wgMLST schemes  45. As the later provides slightly higher discrimination among Bp isolates than the cgMLST_pertussis scheme, it might provide valuable additional genotyping information for local transmission investigations and may be incorporated into the unified platform in the future. We provided examples of applications of the genomic database and its associated genotyping schemes, from the analysis of the genus-level diversity of species, to isolate genotyping within    S3  and S7 ), more genomes of Bppov would be needed to define its phylogenetic origins and unique genomic features.
We also created a genotyping scheme dedicated to macrolide resistance, the possible dissemination of which is an important point of public health concern. To enable easy identification of the macrolide-resistant sublineage that is currently highly prevalent in China, we defined a nomenclature of alleles of the associated marker fhaB, providing a convenient way to distinguish 'out of China' dissemination of the fhaB3 sublineage, from parallel evolution of macrolide resistance in other locations. The following limitations of the present work must be considered. First, there is an imbalance towards B. pertussis genomic sequences from countries that use acellular vaccines. The genomic database currently contains only few genomes of isolates from Africa (0. 2%), Asia (1. 9%) or Oceania (2. 1%) whereas a majority comes from Europe (58.
7%). Still, historical isolates from Europe and the USA, and more recent isolates from China  24, 25, 50  , are available and represent a diversity of sequences from the ptxP1 branches that are typically prevalent in countries using whole-cell vaccines. In the future, efforts should be made to generate and incorporate more genomic sequences from world regions that are currently underrepresented, and the genomic database and attached analytical tools may promote their integrated analysis. A second limitation is that several genotyping schemes are not applicable to the entire breadth of phylogenetic diversity of the database; for example, the T3SS scheme is meant only for genotyping BbGS isolates. More dedicated genotyping schemes can be incorporated in the future to address specific needs, including for population biology studies of the so far little sampled environmental species.
Development of pan-genus Bordetella cgMLST scheme A genus-wide core genome MLST scheme  56  , called Bordetella cgMLST v1. 0, was established alongside a genus-wide locus annotation system (BORD loci), using the principles published for the genus Neisseria  57. Six finished and annotated Bordetella genomes were uploaded into the BIGSdb Oxford database  58  : Bordetella pertussis Tohama I  22  ; Bordetella pertussis CS  59  ; Bordetella bronchiseptica RB50  22  ; Bordetella parapertussis 12822  22  ; Bordetella petri 12804  17  ; and, Bordetella avium 197N  60. Using the GenomeComparator module of BIGSdb, the greatest number of common genes (1469) was found when B. bronchiseptica RB50 was used as the reference genome (even though B. petri 12804 had the greatest number of annotated loci, 5023).
Genes described as 'hypothetical' or 'putative' were removed, resulting in 1415 loci. All loci entered into the database were given a BORD number of the format BORD000000, where the last six digits corresponded to the BB0000 numbers used in the annotation of B. bronchiseptica RB50  22. Consequently, loci in this scheme are in the same order as they appear in the reference genome of B. bronchiseptica RB50. Other annotations and known aliases were included in the locus descriptions for comparison. As with other genus-wide locus schemes, this scheme can be expanded simply by adding additional loci, to give an expandable catalogue of the genus pangenome. We computed the fraction of genome length represented in the cgMLST scheme, by dividing the sum of allele lengths of the called loci, by the genome length, and averaged over all genomes per species (Supplementary Data 2).
Merging of the Oxford and Pasteur databases Two BIGSdb databases were originally designed separately for distinct purposes: while Oxford's PubMLST database offered MLST, Bordetella cgMLST v1. 0 (see above) and virulence genes schemes, the Pasteur database was designed for the sole initial purpose of Bordetella pertussis genotyping, with a Bp-specific cgMLST scheme and a Bp-virulence genes schemes  30. To merge the data available in the two databases, we proceeded as follows. As per BIGSdb dual design, isolates genomes and provenance data were imported into the "isolates" database, whereas allelic definitions of MLST, cgMLST and virulence genes were imported into the "seqdef" database. Regarding the isolates database, we first dumped the Oxford database and uploaded it on the Institut Pasteur server.
Second, we imported Pasteur isolates into this new database. To facilitate the understanding of historical origins of each entry, isolates identification numbers (BIGSdb ID number) were defined as follows: isolates from the original Oxford database were numbered from 1 to 1,914 as in the original database (i. e. , their identification numbers were untouched). Second, the isolates from the original Pasteur database were numbered from 10,000 to 12,214 (original Pasteur identification number + 10000). We also completed the comment fields for these isolates, with the old identification number being added with the suffix word "Pasteur". We added "putative duplicate" in this comment field if the corresponding isolate name was present in both original databases. We also added a "duplicate number" field in the isolates database.
If two or more entries were identified as corresponding to the same isolate, they were attributed the same duplicate number, consecutively. Duplicated data identification was based on a combined analysis of metadata and phylogeny tree reconstruction. At time of merging and closure, the Oxford database comprised 1,914 isolates entries, 57 of which were private, whereas the Pasteur database comprised 2,180 entries, 2,009 of which were private. As of January 7 th , 2022, the platform resulting from the merger comprises 2,581 public isolates entries, and 4,853 isolates in total when considering private entries.
cgMLST schemes Two core genome MLST schemes are available. The first scheme, Bordetella cgMLST v1. 0 (hereafter referred to as cgMLST_genus), was initially defined and hosted in the Oxford platform and was designed to be applicable for the entire Bordetella genus. The second scheme, called cgMLST_pertussis, was originally hosted in the Pasteur database and was built for B. pertussis isolates genotyping only  30. Note that only the latter scheme has attached cgST definitions, i. e. , unique cgST identifiers are attached to each distinct allelic profile. Both cgMLST schemes are available in the merged resource.
Genotyping schemes for vaccine antigens and virulence-associated genes In both original databases, virulence-associated gene schemes had been defined separately. We choose to keep all loci from the virulence scheme designed in Oxford and to add some additional relevant loci. These loci were then grouped into different schemes comprising either Bp vaccine antigens (fim2, fim3, ptxP, ptxA, ptxB, ptxC, ptxD, ptxE, fhaB-2400_5550), T3SS genes (bopB, bopD, bopN, bsp22, bteA), autotransporters (bapC, brkA, tcfA, prn, vag8), other toxins (cyaA, dnt) or phase biology genes (bipA, bvgA, bvgS). We decided to include the prn locus in the autotransporter scheme rather than in the Bp vaccine antigens scheme, because prnnegative isolates would not allow the definition of sequence types (ST) for the vaccine antigens scheme.
To make analyses of virulence-associated and vaccine antigen genes more user-friendly, common gene names used in the literature were used as locus identifier, instead of the locus tags that were initially used in the Oxford database (e. g. , BORD005020 was replaced by fim2 and BORD005021 by fim3). In addition, for five loci common to the original Pasteur and Oxford databases (i. e. fim2, fim3, ptxA, ptxP and tcfA), the allele numbering was re-defined so that the alleles of each locus would match the nomenclature found in the literature for B. pertussis alleles; the correspondence between the original allele numbering system and the new one is defined in Supplementary Data 5. Subsequently, the numbering of alleles was simply incremented as new genomes scans led to novel allele definitions.
Then, all future detection of new alleles were based on 90% identity, 90% alignment parameters using defined type alleles as queries. Criteria for loci associated with virulence were stricter: only new alleles sharing 90% identity, 90% alignment with a type allele and with a complete CDS were recorded (excluding the ptxP promoter locus and the fhaB fragment fhaB-2400_5500). These strict parameters were used to minimize the detection of paralogous sequences and to exclude decaying alleles. For each species, we defined reference strains taken either from literature or from the NCBI RefSeq database (Supplementary Data 4). Type alleles were defined from each reference isolate in order to constrain the search space so as to ensure future consistency of allele definitions. The definition of new alleles was subject to constraining parameters.
Phylogenetic analyses Phylogenetic analyses were performed based on concatenated alignments of individual gene loci defined in the database. To derive a phylogenetic tree based on cgMLST loci, we extracted the amino acid allele sequences of each locus and aligned them with MAFFT v7. 467  62. We then back-translated multiple amino acid sequence alignments to codon alignments and then concatenated them into a supermatrix. We used IQ-TREE v2. 0. 6  63  to infer a phylogenetic tree from this supermatrix of characters with a GTR+G+I evolutionary model and Gubbins (2. 4. 1, default parameters)  63, 64. We assessed branch supports with both bootstrap (1,000 replicates) and aLRT-SH methods  65. Phylogenetic trees were drawn and annotated using iTOL  44. When running Gubbins with cgMLST_genus data, alignments were concatenated following the loci order of the reference genome RB50.
To include the breadth of currently sampled Bordetella diversity, we first downloaded all genomic sequences from public databases and ran a rapid distance mash-based phylogenetic analysis (using Gklust software  66  ). From this, we retained all representative genomes of unique deep branches and down-sampled shallow branches that were represented multiple times. The resulting dataset included the putative novel species initially described as genogroups by Genomic species were defined as groups of isolates that can be differentiated from other such groups using the complete genomic sequence-based ANI cutoff value of at 95%, which corresponds to a classically used threshold value for species delineation  31, 67. ANI values were computed for every possible pairwise comparison using fastANI v 1.