Supplementary Information Deep ocean metagenomes provides insight into the metabolic architecture of bathypelagic microbial communities Authors: Silvia G. Acinas1§*, Pablo Sánchez§1, Guillem Salazar§1,2, Francisco M. Cornejo- Castillo§1,3, Marta Sebastián1,4, Ramiro Logares1, Marta Royo-Llonch1, Lucas Paoli2, Shinichi Sunagawa2, Pascal Hingamp5, Hiroyuki Ogata6, Gipsi Lima-Mendez7,8, Simon Roux9D, José M. González10, Jesús M. Arrieta11, Intikhab S. Alam12, Allan Kamau12, Chris Bowler13,14, Jeroen Raes15,16, Stéphane Pesant17,18, Peer Bork19, Susana Agustí20, Takashi Gojobori12, Dolors Vaqué1, Matthew B. Sullivan21, Carlos Pedrós-Alió22, Ramon Massana1, Carlos M. Duarte23, Josep M. Gasol1,24 FL Taxonomic group Archaea Bacteria Eukaryota undef Viruses 1.00 Relative counts Relative 0.75 0.50 0.25 0.00 PA 1.00 0.75 0.50 0.25 0.00 7 10 13 17 20 23 26 32 35 41 43 50 53 59 62 65 67 74 77 81 82 91 97 103 109 112 118 121 131 134 144 146 Station Supplementary Fig. 1. Relative taxonomic composition at the domain level (Archaea, Bacteria and Eukarya and Viruses) of the M-GeneDB genes found in each of the 58 bathypelagic metagenomes. FL, free-living size fraction (0.2-0.8 µm); PA, particle-attached size fraction (0.8-20 µm). KO count per KEGG metabolism in annotated MP-geneDB genes Count 30000 20000 10000 0 Function unknown Exosome [BR:ko04147] Transporters [BR:ko02000]Enzymes with EC numbers Secretion system [BR:ko02044] ABC transporters [PATH:ko02010] Quorum sensing [PATH:ko02024] Purine metabolism [PATH:ko00230] Ribosome biogenesis [BR:ko03009] Transfer RNA biogenesisPeptidases [BR:ko03016] and inhibitors [BR:ko01002] MitochondrialPyruvate biogenesis metabolism [BR:ko03029] [PATH:ko00620]Bacterial motility proteins [BR:ko02035]DNA replication proteins [BR:ko03032] Two-component system [PATH:ko02020] Amino acid related enzymes [BR:ko01007] Oxidative phosphorylation [PATH:ko00190] Aminoacyl-tRNA biosynthesis [PATH:ko00970] Glycolysis / Gluconeogenesis [PATH:ko00010]Chaperones and folding catalysts [BR:ko03110] Chromosome and associated proteins [BR:ko03036] DNA repair and recombination proteins [BR:ko03400] Cysteine and methionine metabolism [PATH:ko00270] Carbon fixation pathways in prokaryotes [PATH:ko00720] Glyoxylate and dicarboxylateGlycine, serine metabolism and threonine [PATH:ko00630] metabolism [PATH:ko00260] Valine, leucine and isoleucine degradation [PATH:ko00280] Alanine, aspartate and glutamate metabolism [PATH:ko00250] metabolism Supplementary Fig. 2. Histograms showing those genes with a functional annotation based on KEGG metabolism hierarchy III (KO) within the novel genes of the Malaspina Gene DataBase (M-GeneDB). This represent 37% of the novel genes of this catalogue since the rest has no functional annotation and it is only shown for those genes with more than 5000 counts. ota ota Crenarchaeota ota ota ota ota ota ota ota ota ota ota Crenarchaeota ota ota ota ota ota ota ota ota Crenarchaeota ota ota Free-living fraction Particle-attached fraction Supplementary Fig. 3. Relative abundance of protists, prokaryotes, giruses, and viruses in the bathypelagic ocean. a) Protists (small eukaryotes) only in the 0.8-20 µm, b) Prokaryotes (Bacteria and Archaea), c) Giruses and d) Viruses (prophage genes in metagenomes) from the Malaspina bathypelagic metagenomes. Different gene markers or strategies were used to assess the diversity and compute abundances from the metagenomes: i) for protists, the 18S miTags approach was used, ii) for prokaryotes we used clade-specific marker genes from 3,000 reference genomes of Archaea and Bacteria to generate taxonomic abundance profiles in each sample, iii) the marker gene of the Nucleo-cytoplasmic Large DNA Viruses (NCLDV) major capsid was used for giruses and iv) the marker gene of the large subunit of the Terminase (TerL) was used for viruses. The left panel presents the relative abundance of each group per station split into the two size fractions (particle attached, PA, and Free-living, FL). The X-axis shows station (St) and oceans (Atlantic, Indian and Pacific). The right panel presents in pie charts the relative abundances of picoeukaryotes, prokaryotes, giruses and viruses as averages of all the stations. a Size fraction 0.4 ● Free−living Particle−attached ● ● ● Basin 0.2 ● ● ● Agulhas−Cape basin ● ● ● ● ● Brazil basin ● ● ● ● Canary basin ● Fiji basin MDS2 0.0 ● ● Guatemala basin ● ● ● ● ● ● ● ● ● Madagascar basin ● ● ● ● ● ● ● Mid Indian basin ● North American basin ● Out of basins −0.2 ● Pacific basin ● South Australian basin ● −0.50 −0.25 0.00 0.25 0.50 MDS1 b 0.50 Size fraction ● Free−living Particle−attached Basin 0.25 ● Agulhas−Cape basin ● Brazil basin ● ● Canary basin ● ● Fiji basin MDS2 ● Guatemala basin ● ● ● Madagascar basin 0.00 ● ● Mid Indian basin ● ● ● ●● ● ●● ● ● ● North American basin ● ● ● ● ●● ● ● Out of basins ● ● ● ● Pacific basin ● South Australian basin ● ● −0.25 ● −0.2 0.0 0.2 0.4 MDS1 c Size fraction ● Free−living ● Particle−attached ● ● 0.2 ● ● ● Basin ● ● ● ● ● Agulhas−Cape basin ● ● ● Brazil basin ● Canary basin ● ● 0.0 ● ● Fiji basin MDS2 ● ● ● ●● ● ● ●● Guatemala basin ● ● ● ● Madagascar basin ● ● Mid Indian basin ● North American basin −0.2 ● Out of basins ● Pacific basin ● South Australian basin ● −0.4 −0.2 0.0 0.2 0.4 0.6 MDS1 Supplementary Fig. 4. Functional structure of the microbial deep ocean communities. Bray Curtis distances among sites using non-metric multidimensional scaling (NMDS) ordination plots based on the functional abundance tables constructed with A) Protein families (Pfams) and B) Enzyme Commission numbers (ECs) and C) Cluster of Orthologous groups (COGs). Note that in this figure station 62 at 2400 m is included whereas it was excluded from Fig. 2. gi402234835|pmoA|gammaproteobacteria|Uncultured_methane_oxidizing_gamma_proteobacterium gi158632817|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi1026287970|pmoA|gammaproteobacteria|Uncultured_Methylomicrobium_sp. gi963527499|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi402244112|pmoA|gammaproteobacteria|Uncultured_Methylomonas_sp. gi215272884|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi402244193|pmoA|gammaproteobacteria|Uncultured_Methylomonas_sp. gi402244130|pmoA|gammaproteobacteria|Uncultured_Methylomonas_sp. gi402244173|pmoA|gammaproteobacteria|Uncultured_Methylomonas_sp. gi766547614|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi256996998|pmoA|gammaproteobacteria|Uncultured_Methylomonas_sp. gi766547580|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi256997012|pmoA|gammaproteobacteria|Uncultured_Methylomonas_sp. Tree scale: 1 gi158632819|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi190888536|pmoA|gammaproteobacteria|Methylomonas_methanica gi158632993|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacteriumgi166836223|pmoA|gammaproteobacteria|Uncultured_type_I_methanotroph gi158632981|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi766547570|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi158632973|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi15824569|pmoA|gammaproteobacteria|Methylomicrobium_sp. gi402244158|pmoA|gammaproteobacteria|Methylomonas_sp. gi1571085315|pmoA|gammaproteobacteria|Methylomonas_sp. gi959241575|pmoA|gammaproteobacteria|Uncultured_type_I_methanotroph gi1012633735|pmoA|gammaproteobacteria|Methylomarinum_sp. gi158633013|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacteriumgi381344439|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi86604243|pmoA|gammaproteobacteria|Methylosoma_difficile_strain_LC_2 gi766547630|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi156958897|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi158632959|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium KU840814.1|pmoA|alphaproteobacteria|Methanotroph_RSKB_FBF4 KU133552.1|pmoA|Archaea|Uncultured_archaeon gi295855436|pmoA|gammaproteobacteria|Methylovulum_miyakonense KU133553.1|pmoA|Archaea|Uncultured_archaeon gi575798089|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. KU133551.1|pmoA|Archaea|Uncultured_archaeon gi1026287960|pmoA|gammaproteobacteria|Uncultured_type_I_methanotroph gi922061378|pmoA|gammaproteobacteria|Methylovulum_sp. gi575798079|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp.KU840820.1|pmoA|alphaproteobacteria|Methanotroph_RSKB_FBF10KU840815.1|pmoA|alphaproteobacteria|Methanotroph_RSKB_FBF5 gi342731308|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi342731300|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi156958883|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi156958891|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. Taxonomy gi342731318|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi342731316|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi363980918|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi1026287972|pmoA|gammaproteobacteria|Uncultured_type_I_methanotrophgi363980910|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi1026287968|pmoA|gammaproteobacteria|Uncultured_type_I_methanotrophgi363980940|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi170664300|pmoA|gammaproteobacteria|Uncultured_type_I_methanotroph gi215272892|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium Archaea gi166836265|pmoA|gammaproteobacteria|Uncultured_type_I_methanotrophgi258578723|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp. gi766547526|pmoA|gammaproteobacteria|Uncultured_gamma_proteobacterium gi381344453|pmoA|gammaproteobacteria|Uncultured_Methylobacter_sp.