dataesr/scientific-paragraphs-categorization huggingface dataset:
3 , Supplementary Notes). Consistent with the presence of a highly conserved CAAAAA motif, we identified a Type II 6mA solitary DNA MTase (577 aa) present across isolates (Fig. 1f , Supplementary Table  2b , Supplementary Notes) and responsible for methylation of the former. This MTase is encoded by CD2758 in the reference strain C. difficile 630  2, 21. Here we have named CD2758 as CamA (C. difficile adenine methyltransferase A). Its ubiquity was not restricted to the 36 isolates, as we were able to retrieve orthologs in a list of ~300 global C. difficile isolates from GenBank (Supplementary Table  4 ). REBASE also showed functional orthologs of camA only in very few other Clostridiales and Fusobacteriales (Extended Data Fig. 4 ), suggesting that this MTase is fairly unique to C. difficile.
Materials and Methods Clostridioides difficile isolates and culture 36 clonal C. difficile isolates from infected fecal samples were obtained using protocols developed in an ongoing Pathogen Surveillance Program at Mount Sinai Hospital (Supplementary Table  1 ). Additionally, 9 fully sequenced and assembled C. difficile genomes were retrieved from Genbank Refseq (ftp://ftp. ncbi. nih. gov/genomes, last accessed in November 2016) (Supplementary Table  1 ). Raw sequencing data from global and UK collections comprising 291 C. difficile 027/BI/NAPI genomes were used 3 (Supplementary Table  4 ). C. difficile positive stool samples were frozen at -80 °C prior to analysis. All stool samples underwent culture for C. difficile using an ethanol shock culture method  47. Briefly, approximately 80 mg of solid stool (50 μl liquid stool samples) was added to 0.
De novo genome assembly and motif discovery The RS_HGAP3 protocol was used for de novo genome assembly, followed by custom scripts for genome finishing and annotation (https://github. com/powerpak/pathogendbpipeline). RS_Modification_and_Motif_Analysis. 1 was used for de novo methylation motif discovery. A custom script was used to examine each motif to ensure its reliable methylation states. In brief, variations of a putative motif are examined by comparing the ipdR distribution of each variation with non-methylated motifs.
Identification of defense systems Identification of R-M systems was performed as previously described  51. Briefly, curated reference protein sequences of Types I, II, IIC and III R-M systems and Type IV REases were downloaded from the data set 'gold standards' of REBASE  52  (last accessed in November 2016). All-against-all searches were performed for REase and MTase standard protein sequences retrieved from REBASE using BLASTP v2. 5. 0+ (default settings, e value < 10 -3 ). The resulting e values were log-transformed and used for clustering into protein families by Markov Clustering (MCL) v14-137  53. Each protein family was aligned with MAFFT v7. 305b  54  using the E-INS-i option, 1,000 cycles of iterative refinement, and offset 0. Alignments were visualized in SEAVIEW v4. 6. 1  55  and manually trimmed to remove poorly aligned regions at the extremities.
Hidden Markov model (HMM) profiles were then built from each multiple sequence alignment (available at https://github. com/pedrocas81) using the hmmbuild program from the HMMER v3. 0 suite 56 (default parameters). Types I, II, and III R-M systems were identified by searching genes encoding the MTase and REase components at less than five genes apart. CRISPR repeats were identified using the CRISPR Recognition Tool (CRT) v1. 2  57  with default parameters. For CRISPR spacer homology search, we considered as positive hits those with at least 80% identity. For cas gene identification, we obtained Cas protein family HMMs from the TIGRFAM database 58 v15. 0 and PFAM families annotated as Cas families (downloaded from ftp://ftp. ncbi. nih. gov/pub/ wolf/_suppl/CRISPRclass/crisprPro. html). In total we collected 129 known Cas protein families (98 TIGRFAMS and 31 PFAMs), which were used for similarity searching.
Genes pertaining to abortive infection (Abi) systems were searched with the PFAM profiles PF07751, PF08843, and PF14253 (last accessed in January 2018). Bacteriophage Exclusion (BREX) systems were searched using PFAM profiles for the core genes pglZ (PF08655) and brxC/pglY (PF10923), and specific PFAM profiles for each BREX type as indicated previously  59. DISARM systems were identified using the PFAM signature domains (PF09369, PF00271, PF13091) belonging to the core gene triplet characteristic of this system  60. To search for prokaryotic Argonaute (pAgo) genes we built a dedicated HMM profile based on a list of 90 Ago-PIWI proteins  61. Searches for the ensemble of newly found antiphage systems were performed using the list of PFAM profiles published by the authors  62. Type II toxin-antitoxin (T-A) systems were detected using the TAFinder tool  63  with default parameters.
C. difficile strain construction ACE was used to construct 630ΔermΔpyrEΔcamA using uracil and 5-fluoroorotic acid to select for plasmid excision as previously described  83. The flanking primer pair #2274 / 2279 was used to screen for the camA deletion as shown in Extended Data Fig. 5a  (primers are provided in Supplementary Table  5b ). Colonies that appeared to harbor gene deletions were validated by performing an internal PCR using a primer (#2288) that binds within the region deleted and a primer (#2279) that binds to the region flanking the deletion. Two independent clones from the allelic exchange were phenotypically characterized. The camA complementation strains were constructed as previously described by using CDDM plates to select for restoration of the pyrE locus via recombination  83.
Cell length measurements Cells were grown to mid-log and stationary phase in BHIS broth or sporulation was induced as described below for three biological replicates. Cells were imaged using phase contrast microscopy on a Zeiss Axioskop with a 100x Zeiss Plan Neofluar objective (1. 3 NA) at each timepoint. Cell length was calculated using the MicrobeJ plugin for Fiji/ImageJ  84. Image thresholding was done using the local default method in MicrobeJ/Fiji to account for variations in background. Cell detection parameters were optimized (Area: 0-20 μm 2 , Length: 1 μm-max, Width: 0. 5-1 μm) and contours were generated using an interpolated rod-shaped method. Cell length data was exported from MicrobeJ and analyzed using Prism 8 (Graph-pad).
Germination assay Germination assays were performed as previously described  33. Spores (0. 35 OD 600 units, corresponding to ~1 × 10 7 ) were resuspended in 100 μl of water, and 10 μL of this mixture was removed for 10-fold serial dilutions in PBS. The dilutions were plated on BHIS-TA, and colonies arising from germinated spores were enumerated after 18-21 h. Germination efficiencies were calculated by averaging the CFUs produced by spores for a given strain relative to the number produced by wild-type spores for three biological replicates. Statistical significance was determined by performing a one-way ANOVA on natural logtransformed data using Tukey's test. The data were transformed because the use of independent spore preparations resulted in a non-normal distribution. Regardless, no statistical significance in germination efficiency was observed for the mutant and its complements.
Whole-genome multiple alignment and classification of CAAAAA positions Whole-genome multiple alignment of 37 genomes (36 C. difficile isolates and C. difficile 630) was produced by the progressiveMauve program 88 v2. 4. 0 with default parameters. Since progressiveMauve does not rely on annotations to guide the alignment, we first used the Mauve Contig Mover  89  to reorder and reorient draft genome contigs according to the reference genome of C. difficile 630. A core alignment was built after filtering and concatenating locally collinear blocks (LCBs) of size ≥50 bp using the stripSubsetLCBs script (http://darlinglab. org/mauve/snapshots/2015/2015-01-09/linux-x64/). The lower value chosen for LCB size accounts for the specific aim of maximizing the number of orthologous motifs detected. The XMFA output format of Mauve was converted to VCF format using dedicated scripts, and VCFtools 90 was used to parse positional variants (SNPs and indels).
Identification of transcription factor binding sites, and transcription start sites Identification of transcription factor binding sites (TFBS) was performed by retrieving C. difficile 630 regulatory sites in FASTA format from the RegPrecise database  91  (last accessed July 2017). These were converted to PSSMs using in-house developed scripts. This led to a total of 21 PSSMs pertaining to 14 distinct transcription factor families (Supplementary Table  7b ). Matches between these matrices and C. difficile genomes was performed with MAST 92 (default settings). MAST output was filtered on the basis of P value. Hits with P value <10 -9 were considered positive, while hits >10 -5 were considered negative. Hits with intermediate P values were only considered positive if the P value of the hit divided by the P value of the worst positive hit was lower than 100.
For the CcpA, LexA, NrdR, and CodY (which have shorter binding sites), we considered positive those hits with P values <10 -8. Transcription start sites (TSSs) were predicted with Parseq 93 under the 'fast' speed option from multiple RNA-seq datasets (see below). Transcription and breakpoint probabilities were computed using a background expression level threshold of 0. 1 and a score penalty of 0. 05. We kept only high-confidence 5' breakpoint hits, located at a maximum distance of 200 bp from the nearest start codon. A ±5 bp window around the TSS was considered if only one single predicted value was obtained; otherwise we considered an interval delimited by the minimum and maximum values predicted by Parseq.
fa>:2:30:10:8:True, SLIDINGWINDOW:4:15, LEADING:20 TRAILING:20, MINLEN:50). Subsequently, rRNA sequences were filtered from the data set using SortMeRNA 95 v2. 1, based on the SILVA 16s and 23s rRNA databases  96  , and Rfam 5s rRNA database  97. The resulting non-rRNA reads were mapped to the C. difficile 630 reference genome using BWA-MEM v0. 7. 17-r1198  98. The resulting bam files were sorted and indexed with SAMTOOLS, and read assignment was performed with featureCounts 99 v1. 6. 4 (excluding multi-mapping and multi-overlapping reads). A gene was included for differential expression analysis if it had more than one count in all samples. Normalization and differential expression testing were performed using the Bioconductor package DESeq2 v1. 18. 1  100. Genes with a false discovery rate (FDR) < 0.
05 and |log 2 FC| > 1 were called as differentially expressed. Functional classification of genes was performed using the DAVID online database (https://david. ncifcrf. gov)  101. GO annotation terms with a gene count ≥5 and P < 0. 05 (one-tailed Fisher's exact test, FDR corrected) were considered to be significant. The reproducibility of DAVID's functional classification was tested with Blast2GO 102 v5. 2 and Panther 103 v14. Briefly, for Blast2GO, we ran BLASTX searches of the C. difficile 630 genome against the entire GenBank bacterial protein database (as of 09/2018). The output, in XML format, was loaded into Blast2GO, and mapping, annotation and enrichment analysis was performed as indicated (http://docs. blast2go. com/user-manual/quick-start/). For Panther, we downloaded the most recent HMM library (ftp.
pantherdb. org/hmm_scoring/ 13. 1/PANTHER13. 1_hmmscoring. tgz), and annotated our C. difficile 630 protein set with pantherScore2. 1. pl. Both input and background gene lists were formatted to the Panther Generic Mapping File type, as described in the website (http://www. pantherdb. org). To assess the significance of the intersection between multiple datasets of differentially expressed genes (typically observed during C. difficile colonization and infection), we collected gene-expression data from in vivo and in vitro studies  [39] [40] [41]  , in which key factors for gut colonization (e. g. , time post-infection, antibiotic exposure, and spatial structure (planktonic, biofilm growth)) were tested. Differentially expressed genes were called under the same conditions as described above.
Mice were randomly assigned into groups, with two mice assigned to the mock condition and six mice (3 male, 3 female) to each infection condition. The experiment was independently repeated to assess consistency of the data. The data from the experiments were combined for analysis for a total of 12 mice (6 male, 6 female) in each infection condition. Mice were inoculated with 10 5 spores by oral gavage. Mock-inoculated animals were included as controls. Cage changes were performed every 48 h post-inoculation. Fecal samples were collected every 24 h for seven days postinoculation. Dilutions were plated on BHIS-agar containing 0. 1% of the germinant taurocholate to enumerate spores as colony forming units (CFU) per gram of feces. Hamster model-The above strains were tested in Syrian golden hamster strain LVG (Mesocricetus auratus; Charles River Laboratories) as described previously  108.
1% (w/v) crystal violet. After 30 minutes, the biofilms were washed again with PBS, and the crystal violet was solubilized with ethanol. Absorbance was read at 570 nm. Three independent experiments were performed, with each strain assayed in quadruplicate in each experiment. Apart from the complete Type-IB gene cluster (cas1-cas8), we also observed two truncated gene clusters lacking cas1, cas2, and cas4. One of the truncated operons was present across all genomes, while the second was restricted to ST-1 and ST-55. (c) Example of a putative 'defense island' detected in CD_020472 harboring: a Druantia-like system, two T-A systems, two solitary MTases, and one Type I R-M system. The Druantia-like system is similar to the previously reported Type II Druantia systems  62  in the sense that a PF00271 helicase conserved C-terminal domain and DUF1998 (PF09369) are associated with a nearby cytosine methylase.
However, it lacks a PF00270 DEAx box helicase. (d) Genomic context of the sduA gene in CD_22456 pertaining to the newly identified Shedu defense system. The gene is located in an integrative conjugative element (ICE) (Supplementary Table  2d ). (e) Observed/expected (O/E) ratios for co-localized defense systems (maximum of 10 genes apart). Only the most abundant systems were included in the analysis. Expected values were obtained by multiplying the total number of defense systems by the fraction of co-localized defense systems. P values correspond to the Chi-square test. ΔcamA construction, purified spore analyses, broth culture growth, and sporulation kinetics. deviation. (c) Phase-contrast microscopy analyses of sporulating culture samples prior to and after spore purification on a density gradient.
No gross differences in spore morphology were observed between wild type and the MTase mutant. The germination efficiency (G. E. ) of purified spores from the indicated strains is shown below. Scale bar represents 5 μm. Microscopy analyses were performed on three independent spore preparations. (d) Chloroform resistance of purified ΔcamA spores relative to wild type. Spores were treated with 10 % chloroform for 15 min after which spore viability was measured by plating untreated and chloroform-treated spores on media containing germinant and measuring colony forming units. No significant differences in germination efficiency or chloroform resistance were observed. Data are presented as mean ± standard deviation of four independent biological replicates. (e) Heat-resistance (H RES ) efficiencies of sporulating cultures 22 h after sporulation induction were determined relative to wild-type.
Data are presented as mean ± standard deviation. Three independent biological replicates per group were used. *** P < 10 -3 , one-way ANOVA with Tukey's test. Integrative Mobile Elements (IMEs), Integrative Conjugative Elements (ICEs), prophages, and other (within the chromosome). No hits were obtained in plasmids. (f) Immediate genomic context of camA. The example shown (including coordinates) refers to the reference genome of C. difficile 630. + / -signs correspond to the sense and antisense strands respectively. Vertical bars correspond to the distribution of the CAAAAA motif. CD2754: phosphodiesterase with a GGDEF domain (PF00990) and a cache domain (PF02743); ptsI and ptsH belong to a phosphotransferase (PTS) system; CD2757: patatinlike phospholipase (PF01734); CD2758 (camA): Type II MTase; CD2759: Rrf2-type transcriptional regulator; CD2760: phosphodiesterase with a GGDEF domain and a conserved EAL domain (PF00563); CD2761: N-acetylmuramoyl-L-alanine amidase; CD2762: undecaprenyl diphosphate synthase.
The genomic context of camA is largely conserved across strains, located ~25 kb upstream of the S-layer biogenesis locus (Extended Data Figs. 4c, d ). Several of the genes flanking camA (including itself) are part of the C. difficile core-genome (see below), suggesting that they may play biological roles fundamental to C. difficile. CamA modulates sporulation levels in C. difficile. (a) Spore purification efficiencies obtained from sporulating cells (n = 3 independent spore preparations, **P < 10 -2 ; one-way ANOVA and Tukey's test). The spore yield (arbitrary units, a. u. ), was determined by measuring the optical density at 600 nm of the resulting spore preparations and correcting for the volume of re-suspension water. Data are presented as mean ± standard deviation (b) Phase-contrast microscopy after 20 h of sporulation induction.
Relation between gene flux and CRISPR spacer content. Relation between gene flux and CRISPR spacer content. (a) Association between genetic flux (horizontal gene transfer (HGT) and homologous recombination (HR, computed using both ClonalFrameML (CFML) and Geneconv)) and number of CRISPR spacers. The latter were used as proxy of their activity. Data was plotted after excluding very similar ST-1 genomes. The criteria to remove these genomes were based on similarities in R-M content, and gene flux, i. e. , all ST-1 genomes but CD_020475, CD_020474, CD_021026 were removed (n = 26). (b) Same as (a) but considering the complete genome dataset (n = 36). Spearman's rank correlation coefficients (ρ) and associated P values (two-sided) are shown in each graph. Extended Data Fig. 3. Interplay between Type I R-M systems and gene flux in C.
The nine conserved motifs (I-VIII and X) typically found in MTases are highlighted. (b) Phylogenetic tree obtained from the MTase alignment. (c) Phylogenetic tree of the 36 C. difficile strains colored by clade (hypervirulent, human/ animal (HA) associated) and MLST sequence type (ST). Shown is the genomic context of camA across the entire dataset. (d) Expanded view of the region shown in Fig. 1f. The example shown (including coordinates) refers to the reference genome of C. difficile 630. + and -signs correspond to the sense and antisense strands respectively. Vertical bars correspond to the distribution of the CAAAAA motif. Extended Data Fig. 5. ΔcamA construction, purified spore analyses, broth culture growth, and sporulation kinetics.
Data is presented as mean ± standard deviation. * P < 0. 05, ** P < 10 -2 , two-tailed unpaired Student's t-test. Extended Data Fig. 10. Overlap between multiple datasets of differentially expressed (DE) genes Overlap between multiple datasets of differentially expressed (DE) genes. Comparisons were performed between DE genes called in this study for each time point (blue-shaded, n = 1,537) and those obtained from (a) Jenior et al. (black-shaded, n = 971) and (b) Fletcher et al. (gray-shaded, 299). Color intensities of the outermost layer represent the P value significance of the intersections (3,896 genes were used as background). The height of the corresponding bars is proportional to the number of common genes in the intersection (indicated at the top of the bars for pairwise comparisons between the different studies).
Significant overlaps were found between our DE dataset and either (a) genes DE during infection in different murine gut microbiome compositions (P < 10 -6 , one-tailed hypergeometric test implemented in SuperExactTest, Bonferroni adjusted), or (b) DE genes obtained from murine gut isolates at increasing time points after infection (P < 10 -4 , onetailed hypergeometric test implemented in SuperExactTest, Bonferroni adjusted).
Acknowledgements We acknowledge  Dr. Richard J. Roberts  ( New England Biolabs, Inc. USA ) for his help with the prediction of R-M systems and orphan MTases in C. difficile genomes using REBASE Tools and for providing helpful comments. He was originally an author of this manuscript, however, as a staunch supporter of the open access movement, he will not author a paper that is not open access. We also acknowledge  Dr. Eduardo P. C. Rocha  ( Institut Pasteur, Paris, France ) for critical reading and for providing helpful comments. The work was primarily funded by  R01 GM114472  (G. F. ) from the  National Institutes of Health and Icahn Institute for Genomics and Multiscale Biology. In addition, the work was funded by  NIH  grants  R01 AI119145  (H. v. B and A. B. ),  R01 AI22232  (A. S. ) and  R01 AI107029  (R.
Extended Data Fig. 2. 2 Relation between gene flux and CRISPR spacer content. Relation between gene flux and CRISPR spacer content. (a) Association between genetic flux (horizontal gene transfer (HGT) and homologous recombination (HR, computed using both ClonalFrameML (CFML) and Geneconv)) and number of CRISPR spacers. The latter were used as proxy of their activity. Data was plotted after excluding very similar ST-1 genomes. The criteria to remove these genomes were based on similarities in R-M content, and gene flux, i. e. , all ST-1 genomes but CD_020475, CD_020474, CD_021026 were removed (n = 26). (b) Same as (a) but considering the complete genome dataset (n = 36). Spearman's rank correlation coefficients (ρ) and associated P values (two-sided) are shown in each graph. Extended Data Fig.
dakarense sp. nov (580 aa, 63% identity), Peptostreptococcaceae bacterium VA2) and Fusobacteriales (Psychrilyobacter atlanticus DSM 19335) using ClustalX. The nine conserved motifs (I-VIII and X) typically found in MTases are highlighted. (b) Phylogenetic tree obtained from the MTase alignment. (c) Phylogenetic tree of the 36 C. difficile strains colored by clade (hypervirulent, human/ animal (HA) associated) and MLST sequence type (ST). Shown is the genomic context of camA across the entire dataset. (d) Expanded view of the region shown in Fig. 1f. The example shown (including coordinates) refers to the reference genome of C. difficile 630. + and -signs correspond to the sense and antisense strands respectively. Vertical bars correspond to the distribution of the CAAAAA motif. Extended Data Fig.
Motivation: Metagenomic and metatranscriptomic sequencing have become increasingly popular tools for producing massive amounts of short-read data, often used for the reconstruction of draft genomes or the detection of (active) genes in microbial communities. Unfortunately, sequence assemblies of such datasets generally remain a computationally challenging task. Frequently, researchers are only interested in a specific group of organisms or genes; yet, the assembly of multiple datasets only to identify candidate sequences for a specific question is sometimes prohibitively slow, forcing researchers to select a subset of available datasets to address their question. Here we present PhyloMagnet, a workflow to screen metaomics datasets for taxa and genes of interest using gene-centric assembly and phylogenetic placement of sequences.