dataesr/scientific-paragraphs-categorization huggingface dataset:
Sequencing reactions were carried out as described in  Darfeuille et al. (2007)  using PCR amplified fragments. Bioinformatic and statistical NGS data analyses Read pre-processing and alignment Reads were first trimmed of low-quality 3' ends using cutadapt 1. 1 (https://cutadapt. readthedocs. org/) and a base quality threshold of 28 (option '-q 28'). Then, reads having an average base quality lower than 28 were discarded using prinseq-lite 0. 20. 4 ('-min_qual_mean 28';  Schmieder and Edwards, 2011). Read pairs for which both mates passed the quality filtering steps were recovered by means of cmpfastq (http://compbio. brc. iop. kcl. ac. uk/software/cmpfastq. php), and mates were assembled into a single sequence using PANDAseq 2. 9  (Masella et al. , 2012)  run with options '-N -o 30 -O 0 t 0.
6 -A simple_bayesian -C empty'. About 5 million read pairs (combining the three biological replicates) could be assembled for the WT (aapA3/pIsoA3) and pIsoA3* (aapA3/pIsoA3*) samples. These assembled reads were aligned onto the 426-nt reference sequence from H. pylori 26695 by the BWA-SW algorithm of BWA 0. 7. 12  (Li and Durbin, 2009)  run with options '-a 1 -b 3 -q 5 r 2 -z 1' to produce alignments in BAM format. Mapped sequences of length 426 showing a single substitution compared to the reference were then extracted using utilities from the samtools 1. 2  (Li et al. , 2009)  and bamtools 2. 3. 0  (Barnett et al. , 2011)  packages based on the various flags and tags in the BAM files (in particular the CIGAR string and NM tag). This gave a dataset of 1,653,406 WT and 2,559,164 pIsoA3* single-substitution sequences.
Statistical analysis Statistical analyses of the differential distribution of substitutions in the WT and pIsoA3* single-substitution sequences were carried out. To determine whether substitutions were enriched at particular positions in the pIsoA3* compared to WT sequences, a 'positional' analysis was conducted by summing together the counts of all sequences that showed a substitution at a given position, regardless of the identity of the substituted nucleotide. A 'nucleotide-specific' analysis comparing the amount of each individual sequence was also done to determine whether particular nucleotides were enriched at specific positions. As positions +87 and +90 were mutated to inactivate the IsoA3 promoter (see Figure  1B ), for the 'nucleotide-specific' analysis all sequences showing a difference to the reference at one or both of these two positions were excluded from the pIsoA3* and WT datasets (11,319 pIsoA3* and 38,575 WT sequences, respectively), and the pIsoA3* reference was converted back to the WT reference in order to make data comparable between WT and pIsoA3* samples.
05). Bar plots of normalized sequence counts and log2 ratios of fold change were drawn using R 3. 2. 0 (R Core Team, 2015. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; http://www. R-project. org/). Similar 'positional' analyses were also carried out for single-insertion and single-deletion sequences to determine whether insertions or deletions were statistically enriched at particular positions in the pIsoA3* dataset. For the 'positional' analysis, a heatmap and hierarchical tree clustering of samples according to sequence count patterns was also performed. This was based on TMM-normalized (trimmed mean of M values), median-centered, log2-transformed FPKM (fragment per kilobase per million reads mapped) values, computed according to the protocol and tools of  Haas et al.
(2013). The Pearson correlation coefficient was used as distance metric and average linkage was chosen as clustering method (options '-sample_dist sample_cor -sample_cor pearson -sample_clust average' for the 'analyze_diff_expr. pl' utility script). A log2 cut-off of 0 and a p-value cut-off of 1 were set (options '-C 0 P 1') in order to include all sequence positions in the map. The clustering script 'PtR' was manually edited to suppress the clustering by sequence (i. e. rows) and sort the positions by numerical order instead. Depending on the type of statistical analysis, 'position-specific' or 'nucleotide-specific', there were statistically enriched substitutions in absence of antitoxin (padj 0. 05) at 70 or 72 positions within the aapA3/IsoA3 locus, respectively (65 positions in common between the two analyses).
Structural alignment and covariation analysis To obtain further support for the proposed secondary structures, a phylogenetic analysis of nucleotide covariation in sequence alignments was carried out. A set of 107 AapA3 mRNA sequences from 55 Helicobacter genomes available in our T1TAdb database (https://d-lab. arna. cnrs. fr/t1tadb) was retrieved. Significantly mutated sequences (annotated as 'ghost' or with a '*' in T1TAdb) were not considered. In addition, strains corresponding to different isolates of other strains were discarded to avoid redundancy (i. e. Rif1 and Rif2, corresponding to 26695, BM012B and BM012S, corresponding to BM012A, and BM013B corresponding to BM013A). Using MAFFT  (Katoh and Toh, 2008) , a structural alignment was generated for two different transcript lengths (45 and 80 nucleotides) representing respectively the nascent AapA3 transcript extending up to the metastable structure 1 or 2.
Acknowledgements We thank all present and past members of the  ARNA laboratory  for helpful discussions and in particular,  Anaı ¨s Le Rhun  for critical reading of the manuscript. This work was supported by  INSERM   U1212 ,  CNRS UMR 5320 ,  Universitede Bordeaux , and  Agence Nationale de la Recherche  (http:// www. agence-nationale-recherche. fr/)grants  Bactox1  and asSUPYCO. This work was performed in collaboration with the  GeT core facility, Toulouse, France  (http://get. genotoul. fr),and was then supported by  France Genomique National  infrastructure, funded as part of ' Investissement d'avenir' program  managed by  Agence Nationale pour la Recherche  (contract  ANR-10-INBS-09 ). This project has also received funding from the  European Union 's  Horizon 2020 research and innovation programme  under the  Marie Sklodowska-Curie  grant agreement No  642738.
Software, algorithm Cutadapt 1. 1 DOI: 10. 14806/ ej. 17. 1. 200 https://cutadapt. readthedocs. org/ Software, algorithm cmpfastq NIHR Biomedical Research Centre for Mental Health http://compbio. brc. iop. kcl. ac. uk/software/ cmpfastq. php Software, algorithm Prinseq-lite 0. 20. 4 (Schmieder and Edwards, 2011) http://prinseq. sourceforge. net/ Software, algorithm PANDAseq 2. 9 (Masella et al. , 2012) https://github. com/ neufeld/pandaseq Software, algorithm BWA-SW algorithm of BWA 0. 7. 12 (Li and Durbin, 2009) https://sourceforge. net/projects/bio-bwa/ Software, algorithm Samtools 1. 2 (Li et al. , 2009) https://sourceforge. net/projects/samtools/ Software, algorithm Bamtools 2. 3. 0 (Barnett et al. , 2011) https://github. com/ pezmaster31/bamtools Software, algorithm R 3. 2. 0 (R Core Team 2015) http://www.
R-project. org/ Software, algorithm Differential analyses and Hierarchical tree clustering: Trinity 2. 2. 0 (Haas et al. , 2013) http://trinityrnaseq. github. io Software, algorithm Differential analyses: DEseq2 1. 10. 1 (Love et al. , 2014) http://www. bioconductor. org/packages/release/ bioc/html/DESeq2. html Software, algorithm MAFFT 7. 407 (Katoh and Toh, 2008) https://mafft. cbrc. jp/ alignment/software/ source. html.
1% formic acid, 15. 9% water, 84% acetonitrile) in solvent A (0. 1% formic acid, 97. 9% water, 2% acetonitrile). Separation was conducted using a nanocolumn (Acclaim Pep-Map C18, 75 m inner diameter ϫ 50 cm long, 3 m particles, 100 Å pores) at 300 nL/min by applying a gradient of 4 to 55% of solvent B during 90 min for GeLC-MS/MS analyses and during 120 min for shotgun analyses. Data were acquired using Xcalibur 2. 1 software (Thermo Fisher Scientific). The instrument was operated in positive mode in data-dependent mode. Survey full scan MS spectra (from 400 to 1800 m/z) were acquired with a resolution set at 60,000. The 20 most intense ions with charge states Ն 2 were sequentially isolated (isolation width: 2 m/z; 1 microscan) and fragmented using CID mode (energy of 35% and wideband-activation enabled).
Dynamic exclusion was active during 30 s with a repeat count of one. Polydimethylcyclosiloxane (m/z, 445. 1200025) ions were used for internal calibration. The mass spectrometry proteomics data have been submitted to the ProteomeXchange Consortium and are available via the PRIDE partner repository  (24)  with the data set identifiers PXD008046 and 10. 6019/PXD008046. Protein Identification and Data Validation-The reliability between replicates was investigated by comparing chromatograms and spec-tra with Xcalibur 2. 1 software. MS/MS ion searches were performed using Mascot search engine v 2. 3. 2 (Matrix Science, London, UK) via Proteome Discoverer 2. 1 software (ThermoFisher Scientific) against National Center for Biotechnology Information (NCBI) database with Chordata taxonomy (782,473 entries, downloaded in January 2017).
Fragments and parents' tolerances were set at 0. 80 Da and 5 ppm, respectively. The search parameters included trypsin as a protease with two allowed missed cleavages and carbamidomethylcysteine, methionine oxidation and acetylation of N-term protein as variable modifications. Mascot results obtained from the target and decoy databases searches were subjected to Scaffold 4. 8. 2 software (Proteome Software, Portland, OR), and displayed as clusters. Peptide and protein identification and validation were performed using the Peptide and Protein Prophet algorithms, respectively (95. 0% probability, at least two different unique peptides), from three technical replicates. The abundance of identified proteins was estimated by calculating the emPAI using Scaffold Qϩ software (version 4. 4, Proteome Software).
Keratins were not taken into consideration in the subsequent analysis as they might be either contaminant from human skin and/or chicken skin. The list of keratins identified in the analysis is however available (supplemental Data S2). Functional Annotation Using Gene Ontology-Gene ontology terms annotations for biological processes and cellular components category provided by the GO consortium (http://www. geneontology. org/) were investigated using protein database with UniprotKB (http:// www. uniprot. org), and genomic databases with Ensembl (http://www. ensembl. org) and NCBI (https://www. ncbi. nlm. nih. gov). Comparison Between Human and Chicken AF Proteomes-IPI numbers and gene symbols corresponding to the proteins identified in the human AF were retrieved from the publication of Cho et al.
(11) , which integrated all previous studies on the human AF proteome. Earlier versions of the human protein sequences were recovered at the European Bioinformatics Institute website (ftp://ftp. ebi. ac. uk/pub/ databases/IPI/) and updated according to the Ensembl database. Human and chicken gene symbols were compared and for associated genes, protein sequences were aligned using BLAST (https://blast. ncbi. nlm. nih. gov/Blast. cgi) to identify similarity and identity percentages between both species. Human orthologues were systematically checked using Ensembl compara (http://www. ensembl. org) to confirm previous alignments. Phylogenetic Analysis-Phylogenetic branches of emergence were defined in order to determine the moment of appearance of each gene in the tree of life. Eight possible branches for gene birth were considered: Opisthokontas (animals and fungi ϳ 1215 million years ago (MYA)), Bilateria (bilateral animals ϳ 937 MYA), Chordata (chordates ϳ 722 MYA), Vertebrata (vertebrates ϳ 535 MYA), Tetrapoda (tetrapods ϳ 371 MYA), Amniota (amniotes ϳ 296 MYA), Sauropsida (reptiles and birds ϳ 276 MYA), and Mammalia (mammals ϳ 220 MYA).
For all identified proteins, the corresponding Ensembl protein ID was retrieved from the Ensembl database and the related phylogenetic trees were analyzed to highlight the specificity of proteins evolution (http://www. ensembl. org) (25). Eighty-nine trees were studied. To complement the phylogenetic trees, the conservation of synteny was systematically observed using Genomicus (http://www. genomicus. biologie. ens. fr) and Mapviewer (https://www. ncbi. nlm. nih. gov/mapview)  (21, 26). The gene was defined as conserved if both surrounding/adjacent genes were the same. Purification of Heparin Binding Proteins-Heparin-Sepharose chromatography was performed according to manufacturer's instructions. Briefly, 2 ml of beads (Heparin Sepharose 6 Flast Flow, GE Healthcare, Sweden) were loaded onto a polypropylene column (Qiagen, Courtaboeuf, France) and washed with 10 volumes of water and 10 volumes of washing buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.
They were obtained as previously described  (27). Experimental Design and Statistical Rationale-To address our scientific questions, we decided to use a conventional chicken laying strain (ISA Brown, Hendrix Genetics, St Brieuc, France) at 38 weeks of age, that were raised at the UE1295 Pole d'ExpeЈrimentation Avicole de Tours (INRA, F-37380 Nouzilly, France). Eighty fertilized eggs were incubated under standard conditions after a three-day storage at 16 °C and 85% relative humidity to favor synchronization of developmental stages-knowing that conditions and duration of storage prior to incubation negatively impact embryo survival and development. The developmental stages were confirmed using the atlas of chicken developmental stages  (29). Egg weight and eggshell quality were both checked and protein profiles of each individual sample were analyzed by SDS-PAGE (supplemental Data S1, sheets #1 and #2).
Protein sequences were retrieved from Scaffold and blasted to check their GenBank accession numbers (NCBI), and to find corresponding Ensembl and Uniprot IDs. Chicken proteins identified in other species were also searched by BLAST alignments. Functional annotation and search for antibacterial candidates were performed by combining Ensembl, Uniprot and NCBI data on chicken proteins and their orthologues. The identification of antibacterial candidates was completed by investigating protein and peptide antibacterial domains using specific sequence analyzing tools such as InterPro (https://www. ebi. ac. uk/ interpro/). To produce enriched fractions, we defined optimal conditions (volume of samples, volume of heparin-Sepharose beads) depending on the initial concentration of proteins in AF and egg white samples from ED0, ED8, ED11, ED14 or ED16 stages.
All samples (raw, flow-through and eluted fractions) were analyzed by SDS-PAGE. Protein profiles of egg white at ED11 and ED16 were compared with previously published data for experimental validation  (27, 30). For antibacterial assays, at least three independent assays were performed on Listeria monocytogenes and Salmonella enterica Enteritidis strains, using two positive controls (one peptide and one protein) purified from chicken egg white. The phylogenetic analysis and corresponding figures were conducted combining data available in Ensembl databases and in literature.
RESULTS Proteomic Profiling of AF-The global protein profile of AF assessed by SDS-PAGE is completely different when comparing the first half (ED8 -11) to the second half of development (ED12-16), due to the massive transfer of egg white into the AF from ED12 onwards (Fig. 3A ). In this context, to better appreciate the intrinsic protein composition of chicken AF, a bottom-up proteomic study combining two different strategies was conducted on AF samples collected at ED11, the last day before egg white transfer. AF of 40 eggs corresponding to 17 females and 22 males (plus one embryo for which tissue sample was discarded) were collected as detailed under Experimental Procedures. Individual SDS-PAGE profiles (using both Coomassie Brilliant Blue and silver staining, supplemental Data S1, sheet #2), protein concentrations, pH, osmolality values, and spectra of absorbance were determined for each AF sample (supplemental Data S1, sheet #1).
Because no significant differences could be detected between individuals regardless of the sex after integration of all parameters, we constituted a pool representing 12 homogenous samples with equilibrated sex ratio for further analyses by high-resolution mass spectrometry. We performed both in-gel and in-solution independent tryptic digestions followed by nanoLC-MS/MS analysis for protein identification and quantification. For in-gel digestion, a 4 -20% SDS-PAGE gel was cut (Fig. 3B ) and each slice was further processed as mentioned under Experimental Procedures. We filtered data for keratins and other proteins that could reflect sample contamination by experimenters (supplemental Data S2). Ninety-one nonredundant proteins were identified, including 49 for shotgun analysis and 70 for GeLC-MS/MS analysis (Fig.
4 ). Twenty-eight proteins were common to both approaches (Fig. 4 ). The identified proteins sometimes refer to other bird species than Gallus gallus, such as Coturnix japonica (japanese quail) or fish Tachysurus fulvidraco (yellow catfish) (supplemental Data S3), either because the Gallus gallus counterpart is still missing in the genome annotation, or because these specific variants have not yet been identified in the chicken species. Raw files, proteins, peptides scores, information and quantitative values are compiled in supplemental Data S2 and S3. Fig. 4  illustrates the results using log10 of the Exponentially Modified Protein Abundance Index (emPAI), and log10 of the percentage of the protein sequence coverage (supplemental Data S3). According to this graph, a set of ten proteins are very abundant compared with the others.
Out of the 936 proteins identified by Cho et al. (11) , after removal of redundancies and considering the updated human genome assembly (GRCh38. p10, GCA_000001405. 25, Dec 2013), we ended with 842 proteins, plus 81 proteins retrieved from the other studies (11) (supplemental Data S3, sheet #4). This data integration led to 923 proteins that are identified so far in the human AF. When considering gene symbols, 48 proteins were found to be common to both chicken and human AF (Fig. 6 ). We confirmed their orthologous relationship using the Ensembl database. Although they do not share the same gene symbol, LOC107051274 and CAO79236. 1 (immunoglobulins) can be likened to human immunoglobulins G, which are omnipresent in human AF  (11).
Identification of Candidate Proteins Responsible for Antibacterial Activity in AF at ED11 and ED16, by Mass Spectrometry-In parallel to gels prepared for antibacterial assays, an SDS-PAGE performed under the same conditions was stained with Coomassie Brilliant Blue. The superimposition of both the stained SDS-PAGE gel and the gel exhibiting antibacterial activities allowed us to select SDS-PAGE bands (labeled A to H, Fig. 7A  and 7B ), which were susceptible to contain antibacterial proteins or peptides. These bands were analyzed by mass spectrometry at ED11 (band A, Fig. 7A ) and at ED16 (bands B to H, Fig. 7B ), which led to the identification of 29 proteins (Table  II ) (supplemental Data S4). Among the most likely candidates possessing antibacterial activity, we could only find LYZ for AF at ED11.
For the sample at ED16, the most probable molecules are LYZ, but also FIG. 5. Functions of the proteins identified in the chicken amniotic fluid at the 11 th day of incubation (ED11). Using Gene Ontology terms, as described under "Experimental Procedures", 80 genes could be assigned to five major functions whereas the biological function of 10 proteins are still not documented in this database (this latter group includes ovalbumin (OVAL) and ovomucoid (SPINK7), two proteins of high abundance (Table  I , supplemental Data S3).
Some proteolytic activities  (18)  have been however detected in AF at ED11, but, because of the low abundance of the corresponding proteins, the biological significance of these activities in AF remains questionable as compared with the egg yolk, where hydrolytic enzymes are concentrated  (43, 49). The same observation can be made for the faint antibacterial activities detected at ED11 in chicken AF, compared with egg white, which concentrates high amounts of antibacterial proteins such as active lysozyme  (30, 42). These considerations along with the detection of antibacterial activities, corroborates the role of chicken AF in protecting the embryo against a range of potential physical, physicochemical and antibacterial threats. Moreover, it also emphasizes some particularities such as the presence in this fluid of bird-specific proteins involved in embryonic nutrition, eggshell biomineralization and egg defense (OVAL, OVALY, OVALX, OC-116, AvBD11) and likely SPINK7/ovomucoid and SPINK5/ovoinhibitor, recently renamed SPIK7 and SPIK5 in the NCBI Gene database (May 2017) and whose functions are still speculative.
(21)  and phylogenetic analysis using Ensembl (http://www. ensembl. org/index. html). For SPINK5, MEPE and SPINK7 genes, which are marked with a question mark, we cannot confirm the distribution of these genes within amniotes, based on the data currently available in Ensembl databases and despite the synteny analysis of these genes. This issue is discussed in the Results section. reproduction. Additionally, this analysis revealed the presence of 43 proteins that have been uniquely found in the chicken AF as compared with human AF (Fig. 6 ). Nevertheless, the high number of proteins reported for human AF as compared with chicken AF, merits further studies. A comprehensive study of the proteome profiling of the allantoic fluid would probably answer some of these questions, as we might find allantoic fluid-specific proteins whose orthologues have been identified in human AF.
Acknowledgments-We thank  Joe ¨l Delaveau  and  Christophe Rat  ( INRA, UE PEAT 609, F-37380 Nouzilly, France ) for providing fertilized eggs;  Angelina Trotereau  and  Nathalie Winter  ( UMR UR1282  Infectiologie Animale et SantePublique, F-37380 Nouzilly, France) who gave us access to L2 laboratories, which were required to perform antimicrobial assays using bacterial pathogenic strains.
The expanded sample (Table  1  and S1 Table)  analyzed here provides further support for the same variants at the SLC6A14 and SLC26A9 loci (Table  2 , minimum p<2. 2x10 -16 and p = 2. 81x10 -11 in column 'Mega-all', and S3 Table ). Support for CFTR (min p = 2. 34x10 -5 at rs213972; gene-based permutation p = 0. 0001; S2 Fig)  and the association between the apical plasma membrane gene-set and meconium ileus was also observed in this study (permutation p<1x10 -4 in a subset of 5,869 unrelated individuals from the full consortium data and p = 0. 017 in the North American Phase II subgroup alone). The confirmation of multiple contributing genes supports the existence of genetic heterogeneity, indicating that meconium ileus is a phenotype of complex genetic etiology within a mendelian disease.
The rs3788766 SNP is in perfect LD (r 2 = 0. 8 and D' = 1) with rs12710568, which is just 714bp 5' of the SLC6A14 TSS. ChIP-seq evidence  [58]  demonstrates the binding of primed AR (androgen receptor), which has been shown in seven independent studies (GSE58428, GSE84432, GSE56288, GSE28950, GSE56086, GSE65478, GSE32892) and this SNP falls within the androgen receptor binding motif (5'-GGA/TACA[N]NNTGTTCT-3'-SNP position marked in brackets  [59] ) and could potentially contribute to the sex-specific risk differences observed (S4 In contrast to the chromosome 13 and X loci, there are additional genes and tissues that show evidence of colocalization with the GWAS variants (S14 Fig) within 1Mbp of the chromosome 1 locus. As a consequence it is more challenging to pinpoint the responsible gene and tissue from the colocalization evidence alone.