S16). However, none of these bacteria reached significance after correction for multiple-hypothesis testing (Fig. Sterols are rare occurrences in bacteria, although they have been found to be produced by a handful of free-living and host-associated bacteria [68, 87]. New shallow water species of Caribbean Ircinia Nardo, 1833 (Porifera: Irciniidae). To address this question, we performed a comparative analysis of the microbiomes of 11 Ircinia species using whole-metagenomic shotgun sequencing data to investigate three aspects of bacterial symbiont genomesthe redundancy in metabolic pathways across taxa, the evolution of genes involved in pathogenesis, and the nature of selection acting on genes relevant to secondary metabolism. We thank the Yale Center for Genome Analysis for their attentiveness in the metagenomic DNA library preparation and sequencing; the staff of the SeaWulf HPC at SBU, which we used to perform the majority of our analyses; and our funders at Experiment.com. Given that bioinformatic analysis is now the rate limiting factor in genomics, we developed EDGE bioinformatics with a user-friendly interface that allows scientists to perform a number of tailored analyses using many cutting-edge tools. Kuperman AA, Zimmerman A, Hamadia S, Ziv O, Gurevich V, Fichtman B, Gavert N, Straussman R, Rechnitzer H, Barzilay M, Shvalb S, Bornstein J, Ben-Shachar I, Yagel S, Haviv I, Koren O, Deep microbial analysis of multiple placentas shows no evidence for a placental microbiome. The dark gray line marks the density distribution of omega values calculated for non-steroid genes, Plots depicting the proportions of nucleotide variability in CSGs compared to MAG-wide values for bacterial classes that had at least three MAGs with CSGs. Filters 5 and 6 were added to control for contamination that might have been introduced to the samples before their processing in the laboratory. performed CLEM experiments. Nature. You will then receive an email that contains a secure link for resetting your password, If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password. An enrichment of CRISPR and other defense-related features in marine sponge-associated microbial metagenomes. All rights reserved. (2012). Due to the lack of reference genomes, de novo assembly of metagenomics data (short reads) is a beneficial and almost inevitable step for metagenomics analysis (Qin etal., 2010). Sheet Full RdRP - CRISPR matches columns: Number of Spacer matches in NC_009523.1_3781897_3786321_CAS-III-B, Number of Spacer matches in other Roseiflexus sp. Sizes of dots reflect phylotype levels, gradually increasing from species to phylum. and T.A. An average of 16.4 bacterial species were detected in any single breast tumor sample, whereas the average was <9 in all other tumor types (P value <1017 for each tumor type, Wilcoxon rank sum test; Fig. 3, Additional file 4: Fig. In the order that they appear in the CSGs, these genes are delta14-sterol reductase [EC:1.3.1.70] (TM7SF2/ERG24), sterol 14alpha-demethylase [EC:1.14.14.154] (CYP51), and lanosterol synthase [EC:5.4.99.7] (LSS/ERG7). Manage and improve your online marketing. (F) Rarefaction plots for the number of bacterial genera that passed all filters in breast tumor, breast NAT, and breast normal samples. LTA was rarely detected in cancer cells or in CD45+/CD68 immune cells (Fig. Of note, several specific domains recurred frequently during this performance evaluation, and manual examination revealed these to be domains of known repeats. TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations. Tree leaves with existing taxonomic information were identified by mapping (MEGA-BLAST, E-value<1e-30, query coverage 95%, subject coverage 95%, Alignment length>200, Identity 98%, (Alignment_length)/Query_length>0.95) VR1507 sequence set to the latest ICTV data at the time of analysis (July 20, 2021 release of the Virus Metadata Repository (VMR) file, corresponding to MSL36, and available at. Genome reduction and microbe-host interactions drive adaptation of a sulfur-oxidizing bacterium associated with a cold seep sponge. For example, which megahit. Yang Z, Bielawski JP. V.V.D. 3, Additional file 4: Fig. A combined transmembrane topology and signal peptide prediction method. Tropical Ircinia are recognized for their roles in driving ecologically important metabolic processes that impact the coral reef, seagrass, and mangrove environments in which they reside. We also observed a distinct microbiome across subtypes of the same tumor type. Origins and evolution of the global RNA virome. To investigate the potential patterns of horizontal gene transmission (HGT), phylogenetic trees were produced via Bayesian inference for each of the three genes in the CSGs using the codon-aware alignments constructed for the aforementioned CODEML analysis. The sequencing data are available under European Bioinformatics Institute (EBI) submission number PRJEB8920. Sci Rep. 2018;8:8425. A total of 424 new, high-quality bacterial metagenome-assembled genomes (MAGs) were produced for 10 Caribbean Ircinia species, which were evaluated alongside 113 publicly available MAGs sourced from the Pacific species Ircinia ramosa. I. ramosa was also enriched for the two ribose transport system genes rbsB/C and the simple sugar transport system genes ABC.SS.A/P. The authors declare no competing interests. JBK and DEC performed the formal analysis. S1). 1 HKU-BGI Bioinformatics Algorithms Here, we performed a comparative analysis of viral genomes from related clades, identifying instances of genomic modularity, such as fusion of genome segments, rearrangement of proteins, and segmentation of polyproteins. Erwin PM, Thacker RW. All bacteria presented had a false discovery rate (FDR)corrected Q value <0.25. Lastly, to account for other potential sources of medical centerspecific contamination, filter 6 excluded bacteria that were not significantly enriched in a specific tumor type across multiple medical centers. S18 and tables S8 and S9). Steroids and squalene in Methylococcus capsulatus grown on methane. Consequences of stop codon reassignment on protein evolution in ciliates with alternative genetic codes. Yu G, Smith DK, Zhu H, Guan Y, Lam TT-Y. Nucleic Acids Res. We also found enrichment of bacterial functions when comparing breast tumor with NAT samples (table S14). Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. 2017;114:24550. To check if the right version of MitoFinder is actually in your PATH: There are many cases where using a singularity container would be easier than installing MitoFinder from source. Genomes OnLine database (GOLD) v.8: overview and updates. Then, for each contig, we defined the %SD as the ratio between all SD ORFs, and all ORFs with a true start (i.e. Virwani PD, Cai L, Yeung PKK, Qian G, Chen Y, Zhou L, Wong JWH, Wang Y, Ho JWK, Lau KK, Qian PY, Chung SK. Nat Publ Group. Global organization and proposed megataxonomy of the virus world. Despite its advantages, constructing a SdBG efficiently is non-trivial. Thirty-nine samples and 10 controls that had fewer than 1000 normalized reads were discarded from further analysis (materials and methods). Size, taxonomic lineage, genetic code, and motif permutations. Summary: MEGAHIT is a NGS de novo assembler for assembling large and complex metagenomics data in a time- and cost-efficient manner. conceptualized and supervised the project. Phototrophic nutrition and symbiont diversity of two Caribbean sponge cyanobacteria symbioses. Despite the pervasive role of microbiomes in Ircinia biology, it is still unknown how they remain in stable association across tropical species. Reference: MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph To scaffold the contigs generated by MEGAHIT, please use SOAPdenovo-fusion. A.P.C. 1C) and demonstrated a similar spatial distribution (Fig. I. felix and I. ramosa were enriched for both components of the type IV R-M system mcrBC and three dnd sulfur modification genes (dndB-D). Sterols in a psychrophilic methanotroph, Methylosphaera hansonii. 2017;62:178393. Article Of the 384,096 contigs assembled by Megahit 9 1, 2). Bookshelf Aminov RI, Mackie RI. A human homologue of the drosophila toll protein signals activation of adaptive immunity. Proc Natl Acad Sci U S A. On the level of individual bacterial phyla, motility genes were only recovered as being completely depleted in Proteobacteria. Comparative and functional genomics of closteroviruses. Chaban B, Hughes HV, Beeby M. The flagellum in bacterial pathogens: for motility and a whole lot more. Six species (I. ramosa, I. cf. Thoendel M, Jeraldo PR, Greenwood-Quaintance KE, Yao JZ, Chia N, Hanssen AD, et al. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Kelly JB, Carlson DE, Low JS, Rice T, Thacker RW. California Privacy Statement, S. spongiarum as evidenced by 16S rRNA barcoding [10], although it should be noted that some cyanobacterial symbionts of sponges, such as Ca. 09.3.3- LMT-K-712-14-0027 . Thompson KJ, Ingle JN, Tang X, Chia N, Jeraldo PR, Walther-Antonio MR, Kandimalla KK, Johnson S, Yao JZ, Harrington SC, Suman VJ, Wang L, Weinshilboum RL, Boughey JC, Kocher J-P, Nelson H, Goetz MP, Kalari KR, A comprehensive analysis of breast cancer microbiota and host gene expression. Families, involved in breaking the monophyly of the respective phyla (note that a leaf can be both an outlier with respect to its own phylum and an intruder into another phylum), were recorded. MEGAHIT assembles the data as a whole, i.e. of the total microbiome composition per host specimen and was present in every host taxon except for I. strobilina. To improve taxonomic assignment, we used the Ribosomal Database Project (RDP) classifier to augment the Greengenes database by assigning a species-level taxonomy to 380,000 bacterial 16S rRNA sequences that originally lacked such taxonomy (45) (table S3 and materials and methods). 32Department of Surgical Oncology (Surgery C), Sheba Medical Center, Ramat Gan, Israel. Y.I.W. Mycothiol is used by bacteria to detoxify reactive oxygen species (56). 2016;3:196. 2017;2:153342. Here, we devised a computational pipeline for sensitive RNA virus detection suitable for analysis of thousands of metatranscriptomes (. Abiotic conditions drive significant variability in nutrient processing by a common Caribbean sponge, Ircinia felix. First, you can use it to assemble and/or identify mitochondrial-like contigs, then use it in a second step to annotate these particular contigs (option -a) with the corresponding additional options. 2008;53:98696. Complex microbial communities shape the dynamics of various environments. Nucleic Acids Res. On a server with 384GB memory, MEGAHIT took 44.1h, 7 times faster than Minia. Our pipeline did not annotate SdmA and Sdmb, the genes involved in the C-4 demethylation step, although these could be annotated as the hypothetical proteins within the CSGs. Rizk 1A). ColabFold Sergey Ovchinnikov MMseqs2 MSA S1), concurrent with previous reports of this gene and, consequently, the Wood-Ljungdahl pathway being absent from sponge microbiomes [11]. G.F., N.G., and Y.Z. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Reads from 1526 samples and 811 negative controls (DNA extraction controls, 16S 5R PCR controls, and paraffin controls) were computationally combined into long amplicons, using Short MUltiple Regions Framework (SMURF) (44) and the Greengenes database as a reference. Three of these species (I. ramosa, I. cf. Next generation sequencing technologies have offered new opportunities to study metagenomics and understand various microbial communities such as human guts, rumen and soil. The SPAdes genome assembler has become the de facto standard de novo genome assembler for Illumina whole genome sequencing data of bacteria and other small microbes. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Curiously, no genes belonging to the biosynthesis of vancomycin group antibiotics pathway (PATH:ko01055) were enriched in sponge metagenomes, although several of these genes were indeed present in the MAGs of Ircinia symbionts. Combined LPS fluorescence staining and transmission electron microscopy (TEM) imaging of the same cells clearly demonstrated the intracellular localization of bacteria in all four tumors (Fig. Engelberts JP, Robbins SJ, de Goeij JM, Aranda M, Bell SC, Webster NS. Metagenomic Assembly (MEGAHIT and meta-SPAdes) Metagenomic Gene Prediction (FragGeneScan and Prodigal) Functional Analysis Module. To determine which bacteria contribute to the MetaCyc pathways that are enriched in the lung tumors of current smokers, we compared the proportion of all bacterial taxa found in lung tumors of current smokers (n = 100) with those in the tumors of never-smokers (n = 43). mSystems. Overall, our analysis of MetaCyc pathways suggests a connection between the functions of bacteria present in the tumor and their tumor microenvironment. [72] and in a Rhodospirillaceae symbiont of Spongia officinalis [57]. Because the tumor microbiome has a relatively low biomass, contamination of the tumor samples with bacteria or bacterial DNA can be problematic (30, 31).Therefore, it is critical to include multiple measures to avoid, or at least detect, any possible contamination in the Research reported in this publication included work performed in the COH Pathology Research Services Core supported by the National Cancer Institute of the National Institutes of Health under award no. In some taxa (e.g. . Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49. Google Scholar. Tropical members of the sponge genus Ircinia possess highly complex microbiomes that perform a broad spectrum of chemical processes that influence host fitness. Megahit Megahit Megahit Megahit helloMegahit Copyright 2022 Elsevier Inc. except certain content provided by third parties. Can sequence phylogenies safely infer the origin of the global virome?. fungi), it's possible to find mitochondrial genes containing intron(s). 2009;25:20789. The biogenic source of these compounds in sponges is still debated, especially for sterols with 24-C side-chain modifications such as 24-isopropylcholesterol, which is used as a biomarker to date sponge fossils to the Neoproterozoic Era [88]. 2Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel. A PCA of the relative abundances of MAGs showed specimens clustering together by host species (Additional file 2: Fig. 19Pediatric Gastroenterology Institute, Rambam Medical Center, Haifa, Israel. Young, Erik A. Lilleskov, Federico J. Castillo, Francis M. Martin, Gary R. LeCleir, Graeme T. Attwood, Hinsby Cadillo-Quiroz, Holly M. Simon, Ian Hewson, Igor V. Grigoriev, James M. Tiedje, Janet K. Jansson, Janey Lee, Jean S. VanderGheynst, Jeff Dangl, Jeff S. Bowman, Jeffrey L. Blanchard, Jennifer L. Bowen, Jiangbing Xu, Jillian F. Banfield, Jody W. Deming, Joel E. Kostka, John M. Gladden, Josephine Z. Rapp, Joshua Sharpe, Katherine D. McMahon, Kathleen K. Treseder, Kay D. Bidle, Kelly C. Wrighton, Kimberlee Thamatrakoln, Klaus Nusslein, Laura K. Meredith, Lucia Ramirez, Marc Buee, Marcel Huntemann, Marina G. Kalyuzhnaya, Mark P. Waldrop, Matthew B. Sullivan, Matthew O. Schrenk, Matthias Hess, Michael A. Vega, Michelle A. OMalley, Monica Medina, Naomi E. Gilbert, Nathalie Delherbe, Olivia U. Mason, Paul Dijkstra, Peter F. Chuckran, Petr Baldrian, Philippe Constant, Ramunas Stepanauskas, Rebecca A. Daly, Regina Lamendella, Robert J. Gruninger, Robert M. McKay, Samuel Hylander, Sarah L. Lebeis, Sarah P. Esser, Silvia G. Acinas, Steven S. Wilhelm, Steven W. Singer, Susannah S. Tringe, Tanja Woyke, T.B.K. 2016;127:1415. government site. For 105 of the colonies, we could not identify the bacteria at the species level (table S5 and materials and methods). The most numerous phyla in the Caribbean Ircinia metagenomes in terms of MAG richness were Proteobacteria (68 Alphaproteobacteria and 49 Gammaproteobacteria), Chloroflexota (82 MAGs), and Poribacteria (40 MAGs). The top BLASTn hits for representative CSGs. To control for nonspecific staining, IHC-negative controls (no primary antibody) and FISH-negative controls (nonspecific complement probe) were also applied to the samples (figs. Miyake K. Innate recognition of lipopolysaccharide by toll-like receptor 4-MD-2. PubMed The igraph software package for complex network research. The ELP graph was constructed from the Interproscan annotations; all others were constructed using the KO annotations produced using EnrichM. An ultrameterized RdRP tree rooted using reverse transcriptases as an outgroup and visualized with ggtree and ggtreeExtra (. mBio. no pre-processing like partitioning and normalization was needed. Bar chart depicting the percent of MAGs that are found across multiple host species. The sequence from Escherichia coli K-12 substrain MG1655 was used as a reference sequence. Front Microbiol. To characterize the intratumor microbiome, we developed a multiplexed 16S rDNA sequencing protocol that amplifies five short regions along the 16S rRNA gene: the 5R 16S rDNA sequencing method (Fig. Only datasets dominated by prokaryotic sequences (P-dominated) containing at least 10 prokaryotic RNA viruses were considered. NIHMS1645237-supplement-Supplementary_Materials.docx. Isme J. R.W. CD45-positive leukocytes generally exhibited a stronger cytoplasmic bacterial staining by 16S rRNA staining than that exhibited by cancer cells (Fig. 2010;74:41733. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. The omega values were substantially lower for the three genes contained in the CSGs (TM7SF2/ERG24 omega = 0.13, LSS/ERG7 omega = 0.17, CYP51 omega = 0.26) (Fig. copy number in Tara Oceans MAGs)/(avg. Comparisons of sponge populations across the barrier reefs of Australia and Belize: evidence for higher productivity in the Caribbean. Proteobacteria was further split at the class level into Alphaproteobacteria (68 Tara Oceans and 73 Ircinia MAGs) and Gammaproteobacteria (38 Tara Oceans and 58 Ircinia MAGs). (F) Principal coordinate analysis (PCoA) biplot on the Jaccard similarity indexes between bacterial species profiles of the different tissue types. 2022 Nov 3. doi: 10.1038/s41564-022-01252-3. In total, we generated 56,565,928 sequence reads that were de novo-assembled and screened for potential aetiological agents. ColabFold Sergey Ovchinnikov MMseqs2 MSAhttps://github.com/sokrypton/ColabFold, ColabFold Yoshitaka Moriwaki LocalColabFold LocalColabFold ColabFold, AlphaFold ParaFoldAlphaFold2, Installation of colabfold_batch finished , AlphaFold GitHub: https://github.com/deepmind/alphafold, ColabFold GitHub: https://github.com/sokrypton/ColabFold, LocalColabFold GitHub: https://github.com/YoshitakaMo/localcolabfold. A.C. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Each inset demonstrates a low magnification of the entire core. 5A). The letter N marks the cell nucleus. codes that form high-density clades (frequency of alt-code sequences 0.5 and above), Alt code - Genetic code information (empty, "Mito" or "Protist", with asterisk if it belongs to an alt-code clade). 4A and fig. Online ahead of print. Bioinformatics. 2). Across hosts, the metagenomes were depleted in genes relevant to pathogenicity and enriched in eukaryotic-like proteins (ELPs) that likely mimic the hosts molecular patterning. helped with preparations of the manuscript. et al. Notably, MEGAHIT can assemble this dataset with as little as 260GB memory, using 55.3h (Supplementary Section 4). Motility and chemotaxis genes have been found to be depleted in the metagenomes of Lamellodysidea herbaceae (particularly flagellar biosynthesis genes) [64], Petrosia ficiformis, Sarcotragus foetidus, and Aplysina aerophoba [63], in a sulfur-oxidizing gammaproteobacterial symbiont of Suberites sp. deMartel C, Ferlay J, Franceschi S, Vignat J, Bray F, Forman D, Plummer M, Global burden of cancers attributable to infections in 2008: A review and synthetic analysis. The exon annotation is based only on the similarity with the reference. S4). and Minia. De novo sequence assembly requires bioinformatic checking of chimeric sequences. 1978;49:16976. It finished assembling a soil metagenomics dataset with 252Gbps in 44.1 and 99.6h on a single computing node with and without a graphics processing unit, respectively. Based on SdBG, we implemented a multiple k-mer size strategy in MEGAHIT (Peng etal., 2012). per species) (Additional file 6: Table S1). . 5Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. Physical vouchers of the sponge specimens used in this study are deposited with the Smithsonian National Museum of Natural History under the following accession numbers: I. campana: 1641986 and 1641983; I. cf. RS-1 (column Number of Spacer matches in NC_009523.1_3781897_3786321_CAS-III-B), and (ii) high correlation to one of the RdRP-containing segments (column Relative abundance correlation to closest RdRP). All discarded contigs were aggregated and supplemented with manually identified DNA encoded contigs, creating a database of false positives, that was used to further filter the metatranscriptome dataset through exclusion of sequences with producing passable matches to the false positive set. Brown MO, Olagunju BO, Giner J-L, Welander PV. Our data do not establish whether intratumor bacteria play a causal role in the development of cancer or whether their presence simply reflects infections of established tumors (60, 61). The wall time for CPU version of MEGAHIT is 99.4h. Minia does not support multi-threads; it was run with k=31 and min_abundance=2. to reduce computational load, all DNA filtrations searches were run until the first reliable match per query (mmseqs max-accept 1, BLASTn max_target_seqs 1, DIAMOND --max-target-seqs 1). Article CAS Google Scholar Medzhitov R, Preston-Hurlburt P, Janeway CA. Reynolds D, Thomas T. Evolution and function of eukaryotic-like proteins from sponge symbionts. Bioinformatics. Pfam annotations were performed on the protein domains using Interproscan v5.39-77 [33]. 2016;7:990. Mercy-kmers strengthen the contiguity of low-depth regions. Preparation for this dataset differed from the enrichment analysis dataset in that the MAGs underwent dereplication across the ten host species instead of within each host species. Lau JT, Whelan FJ, Herath I, Lee CH, Collins SM, Bercik P, Surette MG, Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling. Variants were called by mapping the cleaned and filtered reads for each metagenomic sample to each MAG using BWA MEM v0.7.17 [37]. 2008;68:416. Assuming that the broad host assignment (plants, animals, or fungi) of viruses can be extended over minor sequence dissimilarity (less than 10%), we identified only 1,038 metatranscriptomic contigs that belonged to the same RvANI90 cluster as viruses from VirusHostDB (. We also identified several enzymatic domains implicated in RNA repair and metabolism, including RtcB-like 3-phosphate RNA ligase (. The first tab (Full RdRP - CRISPR matches) lists all hits (0 or 1 mismatches) identified between selected RNA viruses and CRISPR spacers associated with Roseiflexus sp. The resultant VCF files were also filtered using BCFtools to remove variants that had QUAL scores less than 20 or if read depth was anomalously high [40].
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