dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgements Financial support was provided by the project  Ecología funcional de los bosques andaluces y predicciones sobre sus cambios futuros (For-Change ) ( UCO-FEDER 18 REF 27943 MOD B ), the project  Funcionalidad y servicios ecosistémicos de los bosques andaluces y normarroquíes: relaciones con la diversidad vegetal y edáfica ante el cambio climático  ( P18-RT-3455 ) from  Junta de Andalucía (Spain) , by the  Spanish MEC  project  DIVERBOS  ( CGL2011-30285-C02-02 ) and  ECO-MEDIT  ( CGL2014-53236-R ) and FEDER funds. We thank the  MAPA (Ministerio de Agricultura, Pesca y Alimentación)  and  MITECO (Ministerio de Transición Ecológica ) the access and the open-access availability of the Spanish Forest Inventory (https://www. mapa. gob. es/). CCB is supported by a postdoctoral fellowship of the  Ramón Areces Foundation.
Molecular cloning and phylogenetic analysis Total RNA of S. torazame was extracted using TRIzol Reagent (15,596,026; Thermo Fisher Scientific), and the corresponding cDNA was synthesized using SuperScript IV Reverse Transcriptase (18,091,050; Thermo Fisher Scientific). PCR was performed to amplify fragments of the S. torazame Scleraxis (StScx) gene by LA Taq HS (RR042A; TaKaRa Bio Inc. , Japan) with specific primers designed based on the prospective StScx sequence obtained from Squalomix, the elasmobranch transcriptome database (https://transcriptome. riken. jp/squalomix/) [25]. The PCR fragments were cloned into the pGEM-T Easy vector (A1360; Promega, USA) and sequenced. For phylogenetic analysis, amino acid sequences of orthologous genes from other vertebrate and invertebrate species were compiled from GenBank (http://www.
The sagittal section in Fig. 1d  was reconstructed in silico from serial transverse sections using Avizo software version 8. 0. 1 (Thermo Fisher Scientific). In situ hybridization on paraffin sections for S. torazame Scx (GenBank accession number LC430615) was performed as previously described  [30]. Counterstaining for in situ hybridization was performed with Nuclear Fast Red (Vector Laboratories, USA). Adjacent sections were analyzed to compare gene expression patterns and distributions of fluorescently labeled cells. Sections in brightfield images were imaged with a BX53 microscope (Olympus, Japan) with a DP74 digital camera (Olympus). Fluorescent images were processed by ZEN software (Carl Zeiss), and all images were assembled in Adobe Photoshop (Adobe Systems, USA) as previously described  [31].
temperature, pressure). Studies on human telomeric sequences (TTAGGG repeats), in particular, have revealed that minor changes in the oligonucleotide sequence or in the buffer conditions can alter the structure. At least eight different types of intramolecular G4 topologies were identified to date and some sequences are inher-ently polymorphic  (13, 16, 17). In the absence of external factors (i. e. co-solvents, proteins), the nature and concentration of cations predominantly affect the G4 conformation of a given sequence  (18, 19). A seminal example of this issue is the 22-mer human telomeric sequence (22AG in this manuscript), which has been assigned as a parallel G4 by crystallography in potassium conditions (PDB: 1KF1), an antiparallel G4 by NMR in sodium conditions (PDB: 143D), and a mixture of topologies (hybrid & antiparallel) in circular dichroism of potassium-containing solutions  (20) (21) (22).
Therefore, in order to interpret the MS results in the light of an NMR-derived structure in K + we need to systematically verify by solution spectroscopy (CD, UV melting, and 1 H NMR) that the conformation reported in high salt conditions is also the one present in native MS conditions. The objective of the present study is to build a database of G4 sequences with sufficient stability and validated folds (based on UV melting, circular dichroism and NMR spectroscopies) in 1 mM KCl + 100 mM TMAA assay conditions. This will help short-listing sequences for future MS-based ligand screening studies in terms of structural selectivity.
Circular dichroism (CD) All circular dichroism experiments were performed on a Jasco J-815 spectrophotometer equipped with a JASCO CDF 426S Peltier temperature controller using a quartz cuvette (2 mm path length) at 25 • C. The DNA concentration was 10 M for all the measurements. The scanning range was 220-320 nm with 0. 2 nm data pitch, 2 nm bandwidth, and 0. 5-sec response. For each sample, 3 accumulations were acquired with a scan speed of 50 nm/min, then blankcorrected with the data from the corresponding buffer without DNA. The subtracted spectra were normalized to molar ellipticity coefficient ( ε) according to the following Equation (2): ε = θ 32980 × C × l ( 2 ) where θ = CD signal in millidegrees, C = DNA concentration in mol/L, and l = path length in cm. In KCl solution, theses sequences are polymorphic.
The name is therefore not a PDB code, but follows an internal naming convention. The provided bibliographic references showcase known conformers formed by these sequences (sometimes in presence of a ligand or sodium cations). d 2A5P: PDB id of the mutated (Inosine at 10 th position) sequence. 2MGN: PDB id of the wild type sequence in complex with a PhenDC3 ligand. Tm / • C Tm / • C # K + (MS)/% b CD (1 mM e There is no deposited structure for this sequence. The name is therefore not a PDB code, but follows an internal naming convention.
Electrospray mass spectrometry (ESI-MS) All ESI-MS experiments were performed in negative ion mode on an Agilent 6560 IMS-Q-TOF (Agilent Technologies, Santa Clara, CA, USA) with a dual ESI source and soft tuning conditions  (36). The experiments were performed in ion mobility mode (p He = 3. 89 ± 0. 01 torr, T = 296 ± 1 K). The source gas temperature was 200 • C with fragmentor voltage at 350 V (soft conditions, by default). The injected DNA concentrations were 10 M G-quadruplex in 100 mM TMAA and 1 mM KCl (180 L/h flow rate with a syringe pump). The mass spectra and the arrival time distributions for the 5-ions recorded at a drift voltage of 390 V (drift tube entrance: 600 V; drift tube exit: 210 V) are described herein. A separate paper will describe the full dataset (all charge states, various conditions, conversion to collision cross section distributions and comparison with 3D models).
Data processing, app and database Circular dichroism, UV-melting, NMR, and native ESI-MS data filtering, normalization, fitting, and labeling was performed in g4db, an in-house Shiny application included in the g4dbr package, written in RStudio 1. 3. 1056 (http://www. rstudio. com), running R 4. 1 (https://www. Rproject. org). The database is included in the g4dbr package (https://github. com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4. github. io/G4 database. html; http: //doi. org/10. 5281/zenodo. 4200176). The application documentation is provided in supporting information.
Database composition and building We examined 28 sequences (among which 10 human telomeric variants) for which a specific intramolecular G4 structures had been solved by NMR in high KCl concentration (usually ∼100 mM), with the exception of the two polymorphic sequences 22AG and 21G. The sequences will be referred to by their PDB code as listed in Table  1  (see also Supplementary Tables  S2-S29 ). The CD, 1 H NMR, ESI-MS spectra and UV thermal denaturation profiles are all shown in the supporting information (Supplementary Fig-  ures S1-S168 ). Our goal was to gather diverse topologies from the literature with NMR data in potassium. The published solution structures in potassium include many parallel topologies with type I base stacking (homo stacking), some hybrid topologies (in particular, among human telomeric sequences) with type II stacking (homo and hetero stacking), some antiparallel topologies with two G-quartets (type III stacking, hetero stacking), and one antiparallel topology with 3-quartet but a type II stacking (5YEY) (Figure  1A - E).
There is no documented antiparallel topology with 3quartets and a purely type III stacking in potassium; such structures are documented only in sodium (Figure  1F )  (37). The database was built using an in-house, open-source R package, g4dbr (https://github. com/EricLarG4/g4dbr). Specifically, the g4db function is dedicated to the processing, tidying, storing, visualization, and reporting of CD, 1 H NMR, UV-melting and native MS data from oligonucleotide samples. Although developed for the G4 forming sequences characterized in this manuscript, g4db can be used with any nucleic acid sequence. The long-term goal is to provide open-source tools for the deposition of oligonucleotide biophysics data (raw and processed), while allowing for easy and versatile visualization and reporting. In practice, users can employ the app to visualize a database previously generated by g4db in the database module, or visualize and process raw data, then import it into a new or existing database using the importR module (Supplementary Figure  S169 ).
To read raw data in g4db, it must be first pasted into a templated Excel file provided in the package. Several tools are included in the package for the automated processing of data, and can also be used outside of the database scope. The most notable are: -Data filtering by oligonucleotide name, sequence, topology, buffer, cation, and selective writing to databases; -Labeling of MS spectra performed from user-supplied species names, for which the expected m/z are calculated by the application; -Calculation of molar extinction coefficients at 260 nm from the oligonucleotide sequence, using Equation (  1 ). This can be used independently from g4db, using the epsilon. calculator function (included in the g4dbr package); -Conversion of CD data in mdeg to molar extinction coefficient, using Equation (2); -Determination of folded fraction versus temperature, and T m , from UV-melting data (meltR module), using Equation (3).
This will be released as a standalone application, and its performance will be discussed in a separate publication; -MS noise reduction by intensity filtering, which can be carried out independently with the mass. diet function (included in the package). The processed data is consolidated into. Rda files, which can either be consulted in g4db, or can be loaded in base R for uses outside the application scope. In g4db, the data can be visualized with several customizable plots, and exported in reports in word, pdf, or HTML formats. The reports generated for the oligonucleotides characterized herein are collated in supporting information, and are accessible online as well (https://ericlarg4. github. io/G4 database. html). The use of g4db is described in supporting information (Supplementary Figures  S170--S192 ) and the whole application can be found in the Zenodo repository (http://doi.
5LQG can form two different antiparallel basket-type G4s depending upon K + concentration and pH as shown previously  (44) , and is incompletely folded, which makes it unsuitable for ligand screening in MS. Finally, for 5YEY we observe a major population of [M+2K] n-and mostly one conformation according to 1 H-NMR spectra. In summary, for antiparallel sequences, the best sequences for ligand screening are 2KF8 (antiparallel 2-quartet, basket) and 5YEY (antiparallel 3-quartet, chair). 2KM3 is the only antiparallel 2-quartet with a chair conformation, but is incompletely folded ( 25 = 0. 87). Two frequently used polymorphic sequences (21G, 22AG) are also included in the database. These sequences show mixed conformational topology from CD and broad imino proton signal in 1 H NMR spectra in the ESI-MS buffer as well.
Their mass spectra show both [M+1K] n- and [M+2K] n-(n = 4, 5, 6) stoichiometries. Their ion binding distribution and melting temperatures are very similar to those of 2KF8, indicating significant amounts of 2-quartet topologies in the MS-compatible conditions. Ion mobility spectrometry results are also similar to those of the 2-quartet models (see supporting information Figure  S193 ). Parallel G-quadruplexes. A second group of nontelomeric (mainly promoter sequences) with validated folds are parallel-stranded with type I stacking. Eight sequences with length ranging from 18 to 26 nucleotides are fully folded (>90%) in the ESI-MS conditions. These include two artificial constructs (PDB: 2LK7, 2M4P), two c-myc promoter variants (PDB: 1XAV, 2A5P/2MGN), as well as N-myc (PDB: 2LEE), VEGF (PDB: 2M27), Neisseria gonorrhoeae pilE promoters (PDB: 2LXQ) and the CEB25 human minisatellite (PDB: 2LPW) (Figure  5 ).
In-depth structural analysis for each of the conformers for every sequence in native MS buffer by solution NMR is preferred but was, however, beyond the scope of the manuscript. Overall, we recommend to discard sequences for which a significant amount of unfolded folded species is found ( 1 H NMR not matching with literature, different CD profile, 25 << 1 in UV melting, and high abundance of the 0-K + complex in ESI-MS). 5. Consolidate the data in a way that provides the community with open and easy access to raw and processed data, as well as all necessary contextual elements (e. g. oligonucleotide sequence, extinction coefficient, and concentration, buffer and cation nature and concentration, literature references). Here we developed a database and software suite to automatize the gathering, data treatment, storage, and reporting in a completely open way.
DISCUSSION We here listed sequences with different topology and validated fold (** in Table  1 ) to be used as targets for ligand screening by native MS. We note however that the database is biased towards parallel 3-quartet structures because (1) they are over-represented among the intramolecular structures solved to date in potassium and (2) they are typically more stable and less polymorphic, and thus more likely to be preserved in 1 mM KCl. For non-parallel structures, the best models for ESI-MS screening are variants of the human telomeric sequence, with the known caveat that due to inherent polymorphism, ligand-induced conformational changes can occur even for sequences that have one very predominant fold in absence of ligand  (28, 45, 46). Finding suitable candidates with intramolecular type II & type III folding among non-telomeric sequences for MSbased ligand screening in 1 mM K + remains challenging because there are not many such structures solved already in ∼100 mM K +.
Note that for now we limited our database to intramolecular G-quadruplex forming sequences with all-natural nucleotides. Antiparallel structures with more than two quartets are rare in potassium. Among bimolecular G quadruplex sequences, previous results showed that (12TAG) 2 and (G 4 T 4 G 3 ) 2 are not stable enough in 1 mM K +  (46, 47) , but (dG 4 T 4 G 4 ) 2 and (dG 4 T 3 G 4 ) 2 shows [M+3K] n-as major peaks corresponding to 4 quartet antiparallel conformation while (G 3 T 4 G 4 ) 2 form (M+2K) n- in 1 mM KCl. Whether these structures are the same as in solution NMR studies  (24, 46, 48, 49)  remains to be established. The other possibility for expanding the conformational space is to use ammonium acetate (NH 4 OAc) when there is sufficient evidence of conformation and NH 4 + binding from solution NMR studies  (24).
ACKNOWLEDGEMENTS This work was financially supported by the  European Union  ( H2020-MSCA-IF-2017-799695-CROWDASSAY  and  ERC-2013-CoG-616551-DNAFOLDIMS ), and benefited from access to NMR, MS and  CD at the Plateforme de BioPhysico-Chimie Structurale  of the IECB, which staff is acknowledged for their support. Prof. J. L. Mergny  and his group are acknowledged for access to SAFAS spectrophotometer. The authors acknowledge  Dr Samir Amrane  and  Dr Frédéric Rosu  for useful discussions.
Results Genome assembly, annotation, and comparison. We used a hybrid sequencing approach combining 454 reads, Illumina short reads, and chromosomal contact data (HiC), to obtain a reference genome for Cotesia congregata at a chromosomal scale. First, a 207 Mb high-quality genome (contig N50 = 48. 6 kb, scaffolds N50 = 1. 1 Mb and N90 = 65 kb) was obtained for C. congregata using a combination of mate pair 454 pyrosequencing and Illumina sequencing (Supplementary Data 1). Most of the assembly (86%) consisted of 285 scaffolds of over 100 kb. This genome was then reassembled based on experimentally obtained chromosomal contact maps. The HiC method yielded ten main scaffolds comprising >99% of the previously obtained genome assembly (Supplementary Data 1 and Supplementary Fig. 1 ), and corresponding to the ten chromosomes of C.
congregata  15  (Supplementary Fig. 1 ). In addition, draft genomes of five related Cotesia species-C. rubecula, C. glomerata, C. vestalis, C. flavipes, and C. sesamiae-were sequenced and assembled with Illumina shotgun sequencing reads (Supplementary Data 1) for molecular evolution analyses on homologous genes. They respectively resulted in contig N50 values of 13, 9, 15, 20, and 26 kb and cumulative sizes of 216, 243, 176, 155, and 166 Mb. The genome of C. congregata comprises 48. 7% of repeated DNA sequences including 34. 7% of known transposable elements (TEs) (Supplementary Fig. 2 ). Bracovirus proviral segments included TE sequences that had previously been annotated as bracovirus genes: we revealed that the BV26 gene family corresponded to miniature inverted-repeat transposable elements (MITE) derived from Sola2 elements abundant in the wasp genome (Supplementary Fig.
The dataset included 243 amino acid sequences from C. congregata (blue), 203 sequences from M. demolitor (red), 216 sequences from N. vitripennis (orange), 162 sequences from A. mellifera (yellow). The tree was rooted using the Orco (OR-coreceptor) clade. Circles indicate nodes strongly supported by the approximate likelihood-ratio test (black circles aLRT ≥ 0. 95; white circles 0. 90 ≥ aLRT ≤ 0. 95). The scale bar represents 0. 5 expected amino acid substitutions per site. ORs of the five Hymenoptera species are distributed into 18 OR subfamilies previously described in  18  delineated in gray. B Copy number dynamics of OR (olfaction) P450 (detoxification) and Odv-e66 genes, note that the later are found specifically in bracovirus-associated wasps since they derive from the ancestrally integrated nudivirus.
edu/fastx_toolkit) as described in ref. 56. The C. congregata genome was generated by de novo assembly of 454 reads using GS De Novo Assembler from the Newbler software package v2. 8  57. The consensus was polished using the Illumina data as previously described  58. Gaps were filled using GapCloser module from the SOAPdenovo assembler  59. The genomes of C. sesamiae, C. flavipes, C. glomerata, C. rubecula and C. vestalis were assembled with Velvet v1. 2. 07 57 using the following parameters: velveth k-mer 91 -shortPaired -fastq -separate, velvetg -clean yes and specific adapted values for -exp_cov and -cov_cutoff for each species. Chromosome scale assembly of C. congregata genome Hi-C library preparation. The individual wasps had their gut removed and were immediately suspended after sampling in 30 mL of 1X tris-EDTA buffer and formaldehyde at 3% concentration, then fixed for 1 h.
Ten milliliters of glycine at 2. 5 M was added to the mix for quenching during 20 min. A centrifugation recovered the resulting pellet for -80 °C storage and awaiting further use. The libraries were then prepared and sequenced (2 × 75 bp, paired-end Illumina NextSeq with the first ten bases acting as tags), as previously described  60  using the DpnII enzyme. Read processing and Hi-C map generation. The Hi-C read library was processed and mapped onto DpnII fragments of the reference assembly using HiCbox (available at https://github. com/rkoszul/HiC-Box) with bowtie2 61 on the back-end (option --very-sensitive-local, discarding alignments with mapping quality below 30). Fragments were then filtered according to size and coverage distribution, discarding sequences shorter than 50 bp or below one standard deviation away from the mean coverage.
The process yielded eleven main scaffolds comprising >99% of the bin sequences. Polishing and post-assembly processing. Each scaffold was independently polished by reconstructing the initial contig structure whenever relocations or inversions were found. In addition, previously filtered sequences were reintegrated next to their original neighbors in their initial contig when applicable. The implementation is part of instaGRAAL polishing and available at https://github. com/koszullab/ instaGRAAL (run using the -polishing mode). Assembly validation. We performed the validation with QUAST-LG  63  , an updated version of the QUAST analyzer tailored for large genomes. The initial assembly from Illumina short reads was used as reference. The assessed metrics include genomic feature completeness, Nx and NGx statistics as well as global and local misassemblies.
Genome annotations Transposable element annotation. Genome annotation was first done on the C. congregata reference genome and then exported on the genomes of the five other Cotesia species. First, the transposable element annotation was realized following the REPET pipeline comprising a de novo prediction (TEdenovo) and an annotation using TE libraries (TEannot)  65. This annotation was exported into GFF3 files used as mask for the gene annotation. Automated gene annotation. The automated gene prediction and annotation of C. congregata genome was done using Gmove (https://github. com/institut-degenomique/Gmove) integrating different features based on (i) the mapping of Hymenoptera proteins from all hymenopteran genomes available on NCBI and UniProt Hymenoptera, (ii) the mapping of RNA-Seq data from C.
congregata, C. glomerata, C. vestalis, and C. rubecula (this paper and PRJNA289655, PRJNA485865 PRJNA289731), and (iii) ab initio genes predictions using SNAP  66. The automated annotation of the five other Cotesia species was performed using MAKER  67  using the same features as for the annotation of C. congregata but also including the output automated annotation of C. congregata. Automated gene functional annotation. The functional annotation was performed using blastp from BLAST + v2. 5. 0 68 to compare the C. congregata proteins to the NCBI nonredundant database (from the 29/06/2014). The ten best hits below an evalue of 1e-08 without complexity masking were conserved. Interproscan v5. 13-52. 0 69 was used to analyze protein sequences seeking for known protein domains in the different databases available in Interproscan.
Genome completeness evaluation. The completeness of the genomes and annotations were evaluated using Benchmarking Universal Single-Copy Orthologs BUSCO v3  64  using the insecta_odb9 database composed of 1658 genes. Contigs were searched for similarities against the nonredundant NCBI nucleotide (nt) (release November 2019) and the Uniref90 protein (release November 2019) databases using, respectively, blastn from BLAST + v2. 7. 1  68  and diamond v0. 9. 29. 130  72. For both tasks, e-value cutoff was set to 10 -25. Taxa were inferred according to the highest-scoring matches sum across all hits for each taxonomic rank in the two databases. Sequencing coverage was deduced after mapping Illumina paired reads to the assembly with Bowtie2 v2. 3. 4. 2  61. Contigs were then displayed with Blobtools v1.
1. 1 73 using taxon-annotated-GC-coverage plots. Orthologous genes identification, alignment, and phylogeny. Orthologous genes between all genes annotated in the six Cotesia species and the four outgroups (Microplitis demolitor, Nasonia vitripennis, Apis mellifera and Drosophila melanogaster) were identified using OrthoFinder v1. 14  74. Universal single-copy ortholog genes from BUSCO v3  64  were extracted for the six Cotesia species and the four outgroups, aligned using MAFFT v7. 017  75  and concatenated. The species phylogeny was performed on this alignment composed of 1058 orthologous for a length of 611 kb using PhyML program  76  with the HKY85 substitution model, previously determined by jModelTest v2. 1  77  and the branch support were measured after 1000 bootstrap iterations. The cluster of orthologous genes was used to determine the phylogenetic level of each gene represented in Fig.