Appendix A — Microbe Set Enrichment Analysis (MSEA)
Similar to gene set enrichment analyses for genes (Subramanian et al. 2005), an obvious next step following differential abundance analysis in microbiome studies is to conduct enrichment analysis for microbe sets, known as microbe set enrichment analysis (MSEA) (Kou et al. 2020). Similar to GSEA, the primary goal of MSEA is to detect the modest but coordinated changes in pre-specified sets of related microbial features. Such a set might include all the microbes in a specific pathway or microbial genes that have been shown to be co-regulated based on previously published studies. Like GSEA, MSEA aggregates the per-feature statistics across microbes within a microbe set. This corresponds to the hypothesis that many relevant phenotype differences are manifested by small but consistent changes in a set of features.
Subramanian, Aravind, Pablo Tamayo, Vamsi K Mootha, Sayan Mukherjee, Benjamin L Ebert, Michael A Gillette, Amanda Paulovich, et al. 2005. “Gene Set Enrichment Analysis: A Knowledge-Based Approach for Interpreting Genome-Wide Expression Profiles.” Proceedings of the National Academy of Sciences 102 (43): 15545–50. https://doi.org/10.1073/pnas.0506580102.
Kou, Yan, Xiaomin Xu, Zhengnong Zhu, Lei Dai, and Yan Tan. 2020. “Microbe-Set Enrichment Analysis Facilitates Functional Interpretation of Microbiome Profiling Data.” Scientific Reports 10 (1): 21466. https://doi.org/10.1038/s41598-020-78511-y.
The goal of the MSEA approach is to determine if the members of S (microbe set) are randomly distributed throughout the ranked list of features (L) or primarily found at the top or bottom. We will use the R package gsEasy to conduct the MSEA test described by Subramanian et al. (2005).
A.1 Input data for MSEA using species relative abundance data
In this chapter, we will use the publicly available Inflammatory Bowel Diseases (IBD) microbiome data from the integrative Human Microbiome Project (iHMP) available from the curatedMetagenomicData package (Lloyd-Price et al. 2019). We aim to conduct MSEA analysis based on both taxonomic profiles (species relative abundances) and functional profiles (pathway relative abundances).
Lloyd-Price, Jason, Cesar Arze, Ashwin N. Ananthakrishnan, Melanie Schirmer, Julian Avila-Pacheco, Tiffany W. Poon, Elizabeth Andrews, et al. 2019. “Multi-Omics of the Gut Microbial Ecosystem in Inflammatory Bowel Diseases.” Nature 569 (7758): 655–62. https://doi.org/https://doi.org/10.1038/s41586-019-1237-9.
A.2 Performing the MSEA analysis with species relative abundance data
We will first prepare the input feature table and sample metadata for differential abundance analysis using MaAsLin2 (Mallick et al. 2021). The ranked feature list from the differential abundance analysis serves as an input for the MSEA.
Mallick, Himel, Ali Rahnavard, Lauren J. McIver, Siyuan Ma, Yancong Zhang, Long H. Nguyen, Timothy L. Tickle, et al. 2021. “Multivariable Association Discovery in Population-Scale Meta-Omics Studies.” PLOS Computational Biology 17 (11): e1009442. https://doi.org/https://doi.org/10.1371/journal.pcbi.1009442.
##################
# Load iHMP data #
##################
library(curatedMetagenomicData)
library(dplyr)
se_relative <- sampleMetadata |>
filter(study_name == "HMP_2019_ibdmdb") |>
returnSamples("relative_abundance", rownames = "short")
##########################
# Create sample metadata #
##########################
sample_metadata <-
colData(se_relative) |>
as.data.frame() |> filter(visit_number == 1) |>
dplyr::select(c("age", "disease", "antibiotics_current_use"))
#################
# Set reference #
#################
sample_metadata$disease <- as.factor(sample_metadata$disease)
sample_metadata$disease <- relevel(sample_metadata$disease, 'healthy')
###########################
# Create species features #
###########################
feature_species_t <- as.data.frame(assay(se_relative))
rownames(feature_species_t) <- sub('.*s__', '', rownames(feature_species_t))
##############################
# Subset to baseline samples #
##############################
feature_species <- as.data.frame(t(feature_species_t))
feature_species <- feature_species[rownames(sample_metadata),]
feature_species <- feature_species / 100
rm(feature_species_t); rm(se_relative)In the next step, we will use MaAsLin2 to fit a multivariable regression model for testing the association between microbial species abundance versus IBD diagnosis. The analysis method we use here is “LM”, which is the default setting. We also adjust for age and antibiotic usage, following the original study.
library(Maaslin2)
fit_data = Maaslin2(
input_data = feature_species,
input_metadata = sample_metadata,
normalization = "NONE",
output = "output_species",
fixed_effects = c("disease", "age", "antibiotics_current_use"))
## [1] "Creating output folder"
## [1] "Creating output feature tables folder"
## [1] "Creating output fits folder"
## [1] "Creating output figures folder"
## 2024-11-14 13:24:29.383176 INFO::Writing function arguments to log file
## 2024-11-14 13:24:29.397732 INFO::Verifying options selected are valid
## 2024-11-14 13:24:29.425059 INFO::Determining format of input files
## 2024-11-14 13:24:29.425695 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-11-14 13:24:29.43129 INFO::Formula for fixed effects: expr ~ disease + age + antibiotics_current_use
## 2024-11-14 13:24:29.432035 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
## 2024-11-14 13:24:29.432667 INFO::Filter data based on min abundance and min prevalence
## 2024-11-14 13:24:29.4331 INFO::Total samples in data: 136
## 2024-11-14 13:24:29.433513 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
## 2024-11-14 13:24:29.439148 INFO::Total filtered features: 452
## 2024-11-14 13:24:29.439925 INFO::Filtered feature names from abundance and prevalence filtering: Abiotrophia.sp..HMSC24B09, Acidaminococcus.sp..CAG.542, Acinetobacter.lwoffii, Acinetobacter.ursingii, Actinobaculum.sp..oral.taxon.183, Actinomyces.graevenitzii, Actinomyces.sp..HMSC035G02, Actinomyces.sp..HPA0247, Actinomyces.sp..ICM47, Actinomyces.sp..oral.taxon.180, Actinomyces.sp..oral.taxon.181, Actinomyces.urogenitalis, Actinotignum.timonense, Adlercreutzia.caecimuris, Adlercreutzia.equolifaciens, Aeriscardovia.aeriphila, Aerococcus.urinaeequi, Aeromonas.dhakensis, Aeromonas.hydrophila, Aggregatibacter.aphrophilus, Aggregatibacter.segnis, Aggregatibacter.sp..oral.taxon.458, Alistipes.inops, Alistipes.onderdonkii, Alistipes.timonensis, Allisonella.histaminiformans, Alloprevotella.tannerae, Alloscardovia.omnicolens, Amedibacillus.dolichus, Anaerococcus.lactolyticus, Anaerococcus.vaginalis, Anaerocolumna.aminovalerica, Anaerofilum.sp..An201, Anaerofustis.stercorihominis, Anaeroglobus.geminatus, Anaeromassilibacillus.sp..An172, Anaerosporobacter.mobilis, Anaerostipes.caccae, Anaerostipes.sp..494a, Anaerostipes.sp..992a, Anaerotruncus.sp..CAG.528, Arthrospira.platensis, Atopobium.deltae, Atopobium.minutum, Bacteroidales.bacterium.KA00251, Bacteroides.clarus, Bacteroides.fluxus, Bacteroides.nordii, Bacteroides.oleiciplenus, Bacteroides.sp..43_108, Bacteroides.sp..CAG.144, Bacteroides.sp..CAG.530, Bacteroides.sp..CAG.598, Bacteroides.sp..CAG.633, Bacteroides.sp..CAG.661, Bacteroides.sp..D2, Bacteroides.sp..OM08.11, Bacteroides.stercorirosoris, Bacteroidetes.oral.taxon.274, Bavariicoccus.seileri, Bifidobacterium.angulatum, Bifidobacterium.animalis, Bifidobacterium.asteroides, Bifidobacterium.breve, Bifidobacterium.catenulatum, Bifidobacterium.dentium, Bifidobacterium.pseudolongum, Bifidobacterium.pullorum, Blastocystis.sp..subtype.1, Blautia.argi, Blautia.coccoides, Blautia.hansenii, Blautia.hydrogenotrophica, Blautia.producta, Brachyspira.pilosicoli, Butyricicoccus.pullicaecorum, Butyrivibrio.crossotus, Butyrivibrio.sp..CAG.318, Campylobacter.concisus, Campylobacter.gracilis, Campylobacter.hominis, Campylobacter.showae, Campylobacter.upsaliensis, Campylobacter.ureolyticus, Candidatus.Gastranaerophilales.bacterium, Candidatus.Methanomassiliicoccus.intestinalis, Candidatus.Stoquefichus.sp..KLE1796, Catenibacterium.mitsuokai, Cellulosilyticum.lentocellum, Chlamydia.ibidis, Christensenella.hongkongensis, Christensenella.minuta, Citrobacter.amalonaticus, Citrobacter.braakii, Citrobacter.europaeus, Citrobacter.farmeri, Citrobacter.freundii, Citrobacter.pasteurii, Citrobacter.portucalensis, Citrobacter.werkmanii, Citrobacter.youngae, Cloacibacillus.porcorum, Clostridiales.bacterium.1_7_47FAA, Clostridiales.bacterium.CHKCI006, Clostridioides.difficile, Clostridium.baratii, Clostridium.botulinum, Clostridium.butyricum, Clostridium.cadaveris, Clostridium.celatum, Clostridium.disporicum, Clostridium.neonatale, Clostridium.paraputrificum, Clostridium.perfringens, Clostridium.sp..7_2_43FAA, Clostridium.sp..CAG.167, Clostridium.sp..CAG.242, Clostridium.sp..CAG.253, Clostridium.sp..CAG.411, Clostridium.sp..CAG.413, Clostridium.sp..CAG.590, Clostridium.sp..CAG.678, Clostridium.sp..CAG.964, Clostridium.sp..D5, Clostridium.sp..MSTE9, Clostridium.sp..chh4.2, Clostridium.sporogenes, Comamonas.kerstersii, Coprobacillus.cateniformis, Coprobacter.secundus, Coprobacter.sp., Corynebacterium.accolens, Corynebacterium.amycolatum, Corynebacterium.kroppenstedtii, Corynebacterium.matruchotii, Corynebacterium.oculi, Cronobacter.malonaticus, Cronobacter.sakazakii, Cutibacterium.acnes, Cutibacterium.granulosum, Dellaglioa.algida, Desulfovibrio.fairfieldensis, Desulfovibrio.piger, Desulfovibrionaceae.bacterium, Dialister.micraerophilus, Dialister.pneumosintes, Dialister.sp..CAG.357, Dorea.sp..CAG.317, Dysgonomonas.gadei, Dysgonomonas.mossii, Dysgonomonas.sp..37.18, Eikenella.corrodens, Enhydrobacter.aerosaccus, Enorma.massiliensis, Enterobacter.mori, Enterocloster.asparagiformis, Enterococcus.asini, Enterococcus.avium, Enterococcus.casseliflavus, Enterococcus.dispar, Enterococcus.durans, Enterococcus.faecalis, Enterococcus.faecium, Enterococcus.gallinarum, Enterococcus.hirae, Enterococcus.mundtii, Enterococcus.pseudoavium, Enterococcus.raffinosus, Enterococcus.sp..3H8_DIV0648, Enterococcus.thailandicus, Erysipelothrix.larvae, Escherichia.albertii, Escherichia.marmotae, Eubacteriaceae.bacterium.CHKCI005, Eubacterium.coprostanoligenes, Eubacterium.dolichum.CAG.375, Eubacterium.limosum, Eubacterium.sp..An11, Eubacterium.sp..CAG.180, Eubacterium.sp..CAG.274, Eubacterium.sp..OM08.24, Ezakiella.coagulans, Faecalicatena.orotica, Faecalicoccus.pleomorphus, Faecalitalea.cylindroides, Fannyhessea.vaginae, Finegoldia.magna, Firmicutes.bacterium.CAG.110, Firmicutes.bacterium.CAG.145, Firmicutes.bacterium.CAG.170, Firmicutes.bacterium.CAG.238, Firmicutes.bacterium.CAG.424, Firmicutes.bacterium.CAG.534, Firmicutes.bacterium.CAG.646, Firmicutes.bacterium.CAG.95, Flavonifractor.sp..An10, Flavonifractor.sp..An100, Flavonifractor.sp..An306, Fretibacterium.fastidiosum, Frigoribacterium.sp..Leaf8, Fructilactobacillus.sanfranciscensis, Fusobacterium.equinum, Fusobacterium.gonidiaformans, Fusobacterium.mortiferum, Fusobacterium.naviforme, Fusobacterium.nucleatum, Fusobacterium.periodonticum, Fusobacterium.sp..CAG.439, Fusobacterium.sp..oral.taxon.370, Fusobacterium.ulcerans, Gemella.asaccharolytica, Gemella.haemolysans, Gemella.morbillorum, Gemella.sanguinis, Gemmiger.sp..An50, Gemmiger.sp..An87, Gleimia.europaea, Gordonibacter.pamelaeae, Granulicatella.adiacens, Haemophilus.haemolyticus, Haemophilus.influenzae, Haemophilus.parahaemolyticus, Haemophilus.paraphrohaemolyticus, Haemophilus.pittmaniae, Haemophilus.sputorum, Hafnia.alvei, Hafnia.paralvei, Harryflintia.acetispora, Holdemanella.biformis, Klebsiella.aerogenes, Klebsiella.michiganensis, Klebsiella.oxytoca, Klebsiella.quasipneumoniae, Klebsiella.variicola.CAG.634, Kluyvera.ascorbata, Kluyvera.cryocrescens, Kluyvera.georgiana, Kocuria.palustris, Kosakonia.sacchari, Lachnoclostridium.sp..An118, Lachnoclostridium.sp..An131, Lachnoclostridium.sp..An138, Lachnoclostridium.sp..An14, Lachnoclostridium.sp..An181, Lachnoclostridium.sp..An298, Lachnospiraceae.bacterium.2_1_46FAA, Lachnospiraceae.bacterium.oral.taxon.096, Lacrimispora.celerecrescens, Lacticaseibacillus.rhamnosus, Lactiplantibacillus.plantarum, Lactobacillus.acidophilus, Lactobacillus.amylovorus, Lactobacillus.crispatus, Lactobacillus.delbrueckii, Lactobacillus.gasseri, Lactobacillus.jensenii, Lactobacillus.johnsonii, Lactobacillus.paragasseri, Lactococcus.lactis, Lactococcus.petauri, Lactococcus.piscium, Lactonifactor.longoviformis, Lancefieldella.parvula, Lancefieldella.rimae, Latilactobacillus.sakei, Lawsonella.clevelandensis, Leclercia.adecarboxylata, Leuconostoc.garlicum, Leuconostoc.lactis, Ligilactobacillus.animalis, Ligilactobacillus.ruminis, Ligilactobacillus.salivarius, Limosilactobacillus.fermentum, Limosilactobacillus.mucosae, Limosilactobacillus.oris, Limosilactobacillus.reuteri, Limosilactobacillus.vaginalis, Massilimicrobiota.timonensis, Megamonas.funiformis, Megamonas.funiformis.CAG.377, Megamonas.hypermegale, Megasphaera.elsdenii, Megasphaera.micronuciformis, Megasphaera.sp..DISK.18, Megasphaera.sp..MJR8396C, Methanobrevibacter.smithii, Micrococcus.aloeverae, Micrococcus.luteus, Micrococcus.lylae, Microvirgula.aerodenitrificans, Mitsuokella.jalaludinii, Mitsuokella.multacida, Moraxella.osloensis, Morganella.morganii, Murimonas.intestini, Neisseria.cinerea, Neisseria.flavescens, Neisseria.sp..oral.taxon.014, Neisseria.subflava, Obesumbacterium.proteus, Odoribacter.laneus, Oscillibacter.sp..PC13, Oxalobacter.formigenes, Paenibacillus.macerans, Parabacteroides.goldsteinii, Parabacteroides.johnsonii, Parabacteroides.sp..CAG.409, Pararheinheimera.mesophila, Parvimonas.micra, Parvimonas.sp..KA00067, Parvimonas.sp..oral.taxon.110, Parvimonas.sp..oral.taxon.393, Paucilactobacillus.vaccinostercus, Pediococcus.acidilactici, Pediococcus.pentosaceus, Peptococcus.niger, Peptoniphilus.coxii, Peptoniphilus.duerdenii, Peptoniphilus.harei, Peptoniphilus.lacrimalis, Peptoniphilus.sp..BV3C26, Peptoniphilus.sp..HMSC062D09, Peptostreptococcus.anaerobius, Peptostreptococcus.stomatis, Phascolarctobacterium.sp..CAG.266, Phascolarctobacterium.succinatutens, Phocaeicola.sartorii, Phytobacter.palmae, Porphyromonas.asaccharolytica, Porphyromonas.endodontalis, Porphyromonas.somerae, Porphyromonas.sp..HMSC065F10, Porphyromonas.uenonis, Prevotella.amnii, Prevotella.bergensis, Prevotella.bivia, Prevotella.buccae, Prevotella.buccalis, Prevotella.colorans, Prevotella.corporis, Prevotella.dentalis, Prevotella.denticola, Prevotella.disiens, Prevotella.histicola, Prevotella.intermedia, Prevotella.jejuni, Prevotella.melaninogenica, Prevotella.nigrescens, Prevotella.oralis, Prevotella.oris, Prevotella.oulorum, Prevotella.pallens, Prevotella.salivae, Prevotella.sp..885, Prevotella.sp..AM42.24, Prevotella.sp..CAG.1092, Prevotella.sp..CAG.1185, Prevotella.sp..CAG.279, Prevotella.sp..CAG.520, Prevotella.sp..CAG.891, Prevotella.sp..S7.1.8, Prevotella.stercorea, Prevotella.timonensis, Proteus.hauseri, Proteus.mirabilis, Proteus.penneri, Proteus.vulgaris, Providencia.alcalifaciens, Pseudoflavonifractor.capillosus, Pseudoflavonifractor.sp..An184, Pseudoflavonifractor.sp..An85, Pseudomonas.fragi, Pseudomonas.guguanensis, Raoultella.ornithinolytica, Raoultella.planticola, Rikenella.microfusus, Robinsoniella.sp..RHS, Romboutsia.ilealis, Roseburia.sp..CAG.182, Roseburia.sp..CAG.303, Roseburia.sp..CAG.309, Rothia.mucilaginosa, Ruminococcaceae.bacterium.D5, Ruminococcus.callidus, Ruminococcus.obeum.CAG.39, Ruminococcus.sp..CAG.330, Ruminococcus.sp..CAG.403, Ruminococcus.sp..CAG.488, Ruminococcus.sp..CAG.563, Saccharomyces.cerevisiae, Sanguibacteroides.justesenii, Sarcina.ventriculi, Scardovia.wiggsiae, Schaalia.odontolytica, Schaalia.turicensis, Serratia.liquefaciens, Serratia.marcescens, Sharpea.azabuensis, Shuttleworthia.satelles, Slackia.isoflavoniconvertens, Solobacterium.moorei, Staphylococcus.aureus, Staphylococcus.epidermidis, Staphylococcus.hominis, Streptococcus.agalactiae, Streptococcus.australis, Streptococcus.cristatus, Streptococcus.gallolyticus, Streptococcus.gordonii, Streptococcus.infantis, Streptococcus.lutetiensis, Streptococcus.macedonicus, Streptococcus.milleri, Streptococcus.mitis, Streptococcus.mutans, Streptococcus.oralis, Streptococcus.pasteurianus, Streptococcus.peroris, Streptococcus.salivarius.CAG.79, Streptococcus.sanguinis, Streptococcus.sp..A12, Streptococcus.sp..F0442, Streptococcus.sp..HPH0090, Streptococcus.sp..M334, Streptococcus.thermophilus, Streptococcus.vestibularis, Terrisporobacter.othiniensis, Thermoleophilum.album, Tractidigestivibacter.scatoligenes, Treponema.lecithinolyticum, Turicibacter.sanguinis, Tyzzerella.nexilis, Varibaculum.cambriense, Veillonella.rodentium, Veillonella.rogosae, Veillonella.seminalis, Veillonella.sp..CAG.933, Veillonella.tobetsuensis, Victivallis.vadensis, Weissella.cibaria, Weissella.confusa, Weissella.viridescens, Wohlfahrtiimonas.chitiniclastica, Yersinia.frederiksenii, X.Bacteroides..pectinophilus, X.Butyribacterium..methylotrophicum, X.Clostridium..hylemonae, X.Clostridium..methylpentosum, X.Clostridium..scindens, X.Clostridium..spiroforme, X.Eubacterium..brachy, X.Eubacterium..infirmum
## 2024-11-14 13:24:29.4426 INFO::Total filtered features with variance filtering: 0
## 2024-11-14 13:24:29.443078 INFO::Filtered feature names from variance filtering:
## 2024-11-14 13:24:29.443531 INFO::Running selected normalization method: NONE
## 2024-11-14 13:24:29.444017 INFO::Applying z-score to standardize continuous metadata
## 2024-11-14 13:24:29.448168 INFO::Running selected transform method: LOG
## 2024-11-14 13:24:29.450986 INFO::Running selected analysis method: LM
## 2024-11-14 13:24:29.454546 INFO::Fitting model to feature number 1, Acidaminococcus.intestini
## 2024-11-14 13:24:29.460745 INFO::Fitting model to feature number 2, Agathobaculum.butyriciproducens
## 2024-11-14 13:24:29.463 INFO::Fitting model to feature number 3, Akkermansia.muciniphila
## 2024-11-14 13:24:29.465279 INFO::Fitting model to feature number 4, Alistipes.finegoldii
## 2024-11-14 13:24:29.467532 INFO::Fitting model to feature number 5, Alistipes.indistinctus
## 2024-11-14 13:24:29.469735 INFO::Fitting model to feature number 6, Alistipes.putredinis
## 2024-11-14 13:24:29.471999 INFO::Fitting model to feature number 7, Alistipes.shahii
## 2024-11-14 13:24:29.474209 INFO::Fitting model to feature number 8, Anaerobutyricum.hallii
## 2024-11-14 13:24:29.476468 INFO::Fitting model to feature number 9, Anaeromassilibacillus.sp..An250
## 2024-11-14 13:24:29.478667 INFO::Fitting model to feature number 10, Anaerostipes.hadrus
## 2024-11-14 13:24:29.480949 INFO::Fitting model to feature number 11, Anaerotignum.lactatifermentans
## 2024-11-14 13:24:29.483173 INFO::Fitting model to feature number 12, Anaerotruncus.colihominis
## 2024-11-14 13:24:29.485427 INFO::Fitting model to feature number 13, Bacteroides.caccae
## 2024-11-14 13:24:29.487763 INFO::Fitting model to feature number 14, Bacteroides.cellulosilyticus
## 2024-11-14 13:24:29.490081 INFO::Fitting model to feature number 15, Bacteroides.eggerthii
## 2024-11-14 13:24:29.49234 INFO::Fitting model to feature number 16, Bacteroides.faecis
## 2024-11-14 13:24:29.494552 INFO::Fitting model to feature number 17, Bacteroides.faecis.CAG.32
## 2024-11-14 13:24:29.496793 INFO::Fitting model to feature number 18, Bacteroides.finegoldii
## 2024-11-14 13:24:29.49901 INFO::Fitting model to feature number 19, Bacteroides.fragilis
## 2024-11-14 13:24:29.501228 INFO::Fitting model to feature number 20, Bacteroides.galacturonicus
## 2024-11-14 13:24:29.503451 INFO::Fitting model to feature number 21, Bacteroides.intestinalis
## 2024-11-14 13:24:29.505659 INFO::Fitting model to feature number 22, Bacteroides.ovatus
## 2024-11-14 13:24:29.507911 INFO::Fitting model to feature number 23, Bacteroides.salyersiae
## 2024-11-14 13:24:29.510146 INFO::Fitting model to feature number 24, Bacteroides.stercoris
## 2024-11-14 13:24:29.51239 INFO::Fitting model to feature number 25, Bacteroides.thetaiotaomicron
## 2024-11-14 13:24:29.514594 INFO::Fitting model to feature number 26, Bacteroides.uniformis
## 2024-11-14 13:24:29.516819 INFO::Fitting model to feature number 27, Bacteroides.xylanisolvens
## 2024-11-14 13:24:29.519038 INFO::Fitting model to feature number 28, Barnesiella.intestinihominis
## 2024-11-14 13:24:29.52128 INFO::Fitting model to feature number 29, Bifidobacterium.adolescentis
## 2024-11-14 13:24:29.523508 INFO::Fitting model to feature number 30, Bifidobacterium.bifidum
## 2024-11-14 13:24:29.525712 INFO::Fitting model to feature number 31, Bifidobacterium.longum
## 2024-11-14 13:24:29.527965 INFO::Fitting model to feature number 32, Bifidobacterium.pseudocatenulatum
## 2024-11-14 13:24:29.53017 INFO::Fitting model to feature number 33, Bilophila.wadsworthia
## 2024-11-14 13:24:29.532434 INFO::Fitting model to feature number 34, Blautia.obeum
## 2024-11-14 13:24:29.534645 INFO::Fitting model to feature number 35, Blautia.sp..CAG.257
## 2024-11-14 13:24:29.536915 INFO::Fitting model to feature number 36, Blautia.wexlerae
## 2024-11-14 13:24:29.53912 INFO::Fitting model to feature number 37, Butyricimonas.synergistica
## 2024-11-14 13:24:29.541357 INFO::Fitting model to feature number 38, Butyricimonas.virosa
## 2024-11-14 13:24:29.543619 INFO::Fitting model to feature number 39, Clostridium.bolteae.CAG.59
## 2024-11-14 13:24:29.545826 INFO::Fitting model to feature number 40, Clostridium.sp..CAG.299
## 2024-11-14 13:24:29.548038 INFO::Fitting model to feature number 41, Clostridium.sp..CAG.58
## 2024-11-14 13:24:29.550228 INFO::Fitting model to feature number 42, Collinsella.aerofaciens
## 2024-11-14 13:24:29.55249 INFO::Fitting model to feature number 43, Collinsella.intestinalis
## 2024-11-14 13:24:29.554692 INFO::Fitting model to feature number 44, Coprobacter.fastidiosus
## 2024-11-14 13:24:29.556974 INFO::Fitting model to feature number 45, Coprococcus.catus
## 2024-11-14 13:24:29.559201 INFO::Fitting model to feature number 46, Coprococcus.comes
## 2024-11-14 13:24:29.561457 INFO::Fitting model to feature number 47, Coprococcus.eutactus
## 2024-11-14 13:24:29.563708 INFO::Fitting model to feature number 48, Dialister.invisus
## 2024-11-14 13:24:29.565937 INFO::Fitting model to feature number 49, Dielma.fastidiosa
## 2024-11-14 13:24:29.568253 INFO::Fitting model to feature number 50, Dorea.formicigenerans
## 2024-11-14 13:24:29.570467 INFO::Fitting model to feature number 51, Dorea.longicatena
## 2024-11-14 13:24:29.572694 INFO::Fitting model to feature number 52, Eggerthella.lenta
## 2024-11-14 13:24:29.574918 INFO::Fitting model to feature number 53, Eisenbergiella.massiliensis
## 2024-11-14 13:24:29.577141 INFO::Fitting model to feature number 54, Eisenbergiella.tayi
## 2024-11-14 13:24:29.579314 INFO::Fitting model to feature number 55, Enterocloster.aldenensis
## 2024-11-14 13:24:29.581532 INFO::Fitting model to feature number 56, Enterocloster.bolteae
## 2024-11-14 13:24:29.583776 INFO::Fitting model to feature number 57, Enterocloster.citroniae
## 2024-11-14 13:24:29.585976 INFO::Fitting model to feature number 58, Enterocloster.clostridioformis
## 2024-11-14 13:24:29.58822 INFO::Fitting model to feature number 59, Enterocloster.lavalensis
## 2024-11-14 13:24:29.590462 INFO::Fitting model to feature number 60, Erysipelatoclostridium.ramosum
## 2024-11-14 13:24:29.592718 INFO::Fitting model to feature number 61, Escherichia.coli
## 2024-11-14 13:24:29.594968 INFO::Fitting model to feature number 62, Eubacterium.ramulus
## 2024-11-14 13:24:29.597217 INFO::Fitting model to feature number 63, Eubacterium.sp..CAG.251
## 2024-11-14 13:24:29.599512 INFO::Fitting model to feature number 64, Eubacterium.sp..CAG.38
## 2024-11-14 13:24:29.601725 INFO::Fitting model to feature number 65, Eubacterium.ventriosum
## 2024-11-14 13:24:29.603956 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2024-11-14 13:24:29.606174 INFO::Fitting model to feature number 67, Firmicutes.bacterium.CAG.83
## 2024-11-14 13:24:29.609126 INFO::Fitting model to feature number 68, Firmicutes.bacterium.CAG.94
## 2024-11-14 13:24:29.611355 INFO::Fitting model to feature number 69, Flavonifractor.plautii
## 2024-11-14 13:24:29.61362 INFO::Fitting model to feature number 70, Fusicatenibacter.saccharivorans
## 2024-11-14 13:24:29.615856 INFO::Fitting model to feature number 71, Gemmiger.formicilis
## 2024-11-14 13:24:29.618062 INFO::Fitting model to feature number 72, Haemophilus.parainfluenzae
## 2024-11-14 13:24:29.620302 INFO::Fitting model to feature number 73, Haemophilus.sp..HMSC71H05
## 2024-11-14 13:24:29.622549 INFO::Fitting model to feature number 74, Holdemania.filiformis
## 2024-11-14 13:24:29.624793 INFO::Fitting model to feature number 75, Hungatella.hathewayi
## 2024-11-14 13:24:29.627004 INFO::Fitting model to feature number 76, Intestinibacter.bartlettii
## 2024-11-14 13:24:29.629297 INFO::Fitting model to feature number 77, Intestinimonas.butyriciproducens
## 2024-11-14 13:24:29.631623 INFO::Fitting model to feature number 78, Klebsiella.pneumoniae
## 2024-11-14 13:24:29.633917 INFO::Fitting model to feature number 79, Klebsiella.variicola
## 2024-11-14 13:24:29.636214 INFO::Fitting model to feature number 80, Lachnospira.eligens
## 2024-11-14 13:24:29.63849 INFO::Fitting model to feature number 81, Lachnospira.pectinoschiza
## 2024-11-14 13:24:29.64075 INFO::Fitting model to feature number 82, Lacrimispora.saccharolytica
## 2024-11-14 13:24:29.643019 INFO::Fitting model to feature number 83, Lactobacillus.rogosae
## 2024-11-14 13:24:29.64532 INFO::Fitting model to feature number 84, Lawsonibacter.asaccharolyticus
## 2024-11-14 13:24:29.647719 INFO::Fitting model to feature number 85, Monoglobus.pectinilyticus
## 2024-11-14 13:24:29.650139 INFO::Fitting model to feature number 86, Odoribacter.splanchnicus
## 2024-11-14 13:24:29.653215 INFO::Fitting model to feature number 87, Oscillibacter.sp..57_20
## 2024-11-14 13:24:29.65627 INFO::Fitting model to feature number 88, Oscillibacter.sp..CAG.241
## 2024-11-14 13:24:29.659271 INFO::Fitting model to feature number 89, Parabacteroides.distasonis
## 2024-11-14 13:24:29.662317 INFO::Fitting model to feature number 90, Parabacteroides.merdae
## 2024-11-14 13:24:29.665397 INFO::Fitting model to feature number 91, Paraprevotella.clara
## 2024-11-14 13:24:29.668404 INFO::Fitting model to feature number 92, Paraprevotella.xylaniphila
## 2024-11-14 13:24:29.670684 INFO::Fitting model to feature number 93, Parasutterella.excrementihominis
## 2024-11-14 13:24:29.672996 INFO::Fitting model to feature number 94, Phascolarctobacterium.faecium
## 2024-11-14 13:24:29.675765 INFO::Fitting model to feature number 95, Phocaeicola.coprocola
## 2024-11-14 13:24:29.677999 INFO::Fitting model to feature number 96, Phocaeicola.dorei
## 2024-11-14 13:24:29.680275 INFO::Fitting model to feature number 97, Phocaeicola.massiliensis
## 2024-11-14 13:24:29.682493 INFO::Fitting model to feature number 98, Phocaeicola.plebeius
## 2024-11-14 13:24:29.68475 INFO::Fitting model to feature number 99, Phocaeicola.vulgatus
## 2024-11-14 13:24:29.686973 INFO::Fitting model to feature number 100, Prevotella.copri
## 2024-11-14 13:24:29.689297 INFO::Fitting model to feature number 101, Proteobacteria.bacterium.CAG.139
## 2024-11-14 13:24:29.691571 INFO::Fitting model to feature number 102, Roseburia.faecis
## 2024-11-14 13:24:29.69388 INFO::Fitting model to feature number 103, Roseburia.hominis
## 2024-11-14 13:24:29.696159 INFO::Fitting model to feature number 104, Roseburia.intestinalis
## 2024-11-14 13:24:29.698384 INFO::Fitting model to feature number 105, Roseburia.inulinivorans
## 2024-11-14 13:24:29.700646 INFO::Fitting model to feature number 106, Roseburia.sp..CAG.471
## 2024-11-14 13:24:29.702881 INFO::Fitting model to feature number 107, Ruminococcaceae.bacterium.D16
## 2024-11-14 13:24:29.705118 INFO::Fitting model to feature number 108, Ruminococcus.bicirculans
## 2024-11-14 13:24:29.707326 INFO::Fitting model to feature number 109, Ruminococcus.bromii
## 2024-11-14 13:24:29.709661 INFO::Fitting model to feature number 110, Ruthenibacterium.lactatiformans
## 2024-11-14 13:24:29.711901 INFO::Fitting model to feature number 111, Sellimonas.intestinalis
## 2024-11-14 13:24:29.714087 INFO::Fitting model to feature number 112, Streptococcus.parasanguinis
## 2024-11-14 13:24:29.716297 INFO::Fitting model to feature number 113, Streptococcus.salivarius
## 2024-11-14 13:24:29.718515 INFO::Fitting model to feature number 114, Turicimonas.muris
## 2024-11-14 13:24:29.720748 INFO::Fitting model to feature number 115, Veillonella.atypica
## 2024-11-14 13:24:29.722919 INFO::Fitting model to feature number 116, Veillonella.dispar
## 2024-11-14 13:24:29.725131 INFO::Fitting model to feature number 117, Veillonella.infantium
## 2024-11-14 13:24:29.727305 INFO::Fitting model to feature number 118, Veillonella.parvula
## 2024-11-14 13:24:29.729531 INFO::Fitting model to feature number 119, Veillonella.sp..T11011.6
## 2024-11-14 13:24:29.731747 INFO::Fitting model to feature number 120, X.Clostridium..innocuum
## 2024-11-14 13:24:29.733935 INFO::Fitting model to feature number 121, X.Clostridium..leptum
## 2024-11-14 13:24:29.736196 INFO::Fitting model to feature number 122, X.Clostridium..symbiosum
## 2024-11-14 13:24:29.738388 INFO::Fitting model to feature number 123, X.Eubacterium..rectale
## 2024-11-14 13:24:29.740621 INFO::Fitting model to feature number 124, X.Eubacterium..siraeum
## 2024-11-14 13:24:29.74282 INFO::Fitting model to feature number 125, X.Ruminococcus..gnavus
## 2024-11-14 13:24:29.745029 INFO::Fitting model to feature number 126, X.Ruminococcus..lactaris
## 2024-11-14 13:24:29.747227 INFO::Fitting model to feature number 127, X.Ruminococcus..torques
## 2024-11-14 13:24:29.76697 INFO::Counting total values for each feature
## 2024-11-14 13:24:29.778388 INFO::Writing filtered data to file output_species/features/filtered_data.tsv
## 2024-11-14 13:24:29.790814 INFO::Writing filtered, normalized data to file output_species/features/filtered_data_norm.tsv
## 2024-11-14 13:24:29.803484 INFO::Writing filtered, normalized, transformed data to file output_species/features/filtered_data_norm_transformed.tsv
## 2024-11-14 13:24:29.820758 INFO::Writing residuals to file output_species/fits/residuals.rds
## 2024-11-14 13:24:29.826741 INFO::Writing fitted values to file output_species/fits/fitted.rds
## 2024-11-14 13:24:29.832001 INFO::Writing all results to file (ordered by increasing q-values): output_species/all_results.tsv
## 2024-11-14 13:24:29.83478 INFO::Writing the significant results (those which are less than or equal to the threshold of 0.250000 ) to file (ordered by increasing q-values): output_species/significant_results.tsv
## 2024-11-14 13:24:29.835621 INFO::Writing heatmap of significant results to file: output_species/heatmap.pdf
## 2024-11-14 13:24:29.949567 INFO::Writing association plots (one for each significant association) to output folder: output_species
## 2024-11-14 13:24:29.952273 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-11-14 13:24:29.952804 INFO::Plotting data for metadata number 1, age
## 2024-11-14 13:24:29.953705 INFO::Creating scatter plot for continuous data, age vs Alistipes.indistinctus
## 2024-11-14 13:24:30.180451 INFO::Creating scatter plot for continuous data, age vs Lacrimispora.saccharolytica
## 2024-11-14 13:24:30.350969 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bicirculans
## 2024-11-14 13:24:30.47023 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
## 2024-11-14 13:24:30.589679 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
## 2024-11-14 13:24:30.725197 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
## 2024-11-14 13:24:30.85348 INFO::Creating scatter plot for continuous data, age vs Lachnospira.eligens
## 2024-11-14 13:24:30.972772 INFO::Creating scatter plot for continuous data, age vs X.Ruminococcus..gnavus
## 2024-11-14 13:24:31.093357 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.virosa
## 2024-11-14 13:24:31.213278 INFO::Creating scatter plot for continuous data, age vs Lawsonibacter.asaccharolyticus
## 2024-11-14 13:24:31.372742 INFO::Creating scatter plot for continuous data, age vs X.Eubacterium..siraeum
## 2024-11-14 13:24:31.491211 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.synergistica
## 2024-11-14 13:24:31.610303 INFO::Creating scatter plot for continuous data, age vs Monoglobus.pectinilyticus
## 2024-11-14 13:24:31.731088 INFO::Creating scatter plot for continuous data, age vs Veillonella.atypica
## 2024-11-14 13:24:33.289314 INFO::Plotting data for metadata number 2, disease
## 2024-11-14 13:24:33.290441 INFO::Creating boxplot for categorical data, disease vs Alistipes.putredinis
## 2024-11-14 13:24:33.439684 INFO::Creating boxplot for categorical data, disease vs Gemmiger.formicilis
## 2024-11-14 13:24:33.577261 INFO::Creating boxplot for categorical data, disease vs X.Ruminococcus..torques
## 2024-11-14 13:24:33.685394 INFO::Creating boxplot for categorical data, disease vs Ruminococcus.bicirculans
## 2024-11-14 13:24:33.799605 INFO::Creating boxplot for categorical data, disease vs Sellimonas.intestinalis
## 2024-11-14 13:24:33.908711 INFO::Creating boxplot for categorical data, disease vs X.Clostridium..leptum
## 2024-11-14 13:24:34.018213 INFO::Creating boxplot for categorical data, disease vs Alistipes.shahii
## 2024-11-14 13:24:35.080107 INFO::Plotting data for metadata number 3, antibiotics_current_use
## 2024-11-14 13:24:35.081266 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Coprobacter.fastidiosus
## 2024-11-14 13:24:35.188195 INFO::Creating boxplot for categorical data, antibiotics_current_use vs X.Eubacterium..rectale
## 2024-11-14 13:24:35.296458 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Agathobaculum.butyriciproducensUnlike gene expression studies, we do not have well-defined signatures or modules for microbiome data. Here, we will construct data-driven modules using weighted gene co-expression network analysis (WGCNA) (Langfelder and Horvath 2008), (Geistlinger et al. 2023). We aim to ensure that the effect of disease and other covariates has been removed by working on the residuals. Following the WGCNA tutorial, our first step will be to check whether there are any outliers in our data.
Langfelder, Peter, and Steve Horvath. 2008. “WGCNA: An r Package for Weighted Correlation Network Analysis.” BMC Bioinformatics 9: 559. https://doi.org/https://doi.org/10.1186/1471-2105-9-559.
Geistlinger, Ludwig, Chloe Mirzayi, Fatima Zohra, Rimsha Azhar, Shaimaa Elsafoury, Clare Grieve, Jennifer Wokaty, et al. 2023. “BugSigDB Captures Patterns of Differential Abundance Across a Broad Range of Host-Associated Microbial Signatures.” Nature Biotechnology 42 (5): 790–802. https://doi.org/https://doi.org/10.1038/s41587-023-01872-y.
library(WGCNA)
datExpr <- as.data.frame(t(fit_data$residuals))
gsg = goodSamplesGenes(datExpr, verbose = 3)
## Flagging genes and samples with too many missing values...
## ..step 1
## ..Excluding 6 samples from the calculation due to too many missing genes.
## ..step 2
gsg$allOK
## [1] FALSEIf the last statement returns TRUE, no outliers are identified. If not, we need to remove the outliers from the data.
if (!gsg$allOK)
{if (sum(!gsg$goodGenes) > 0)
printFlush(paste(
"Removing genes:",
paste(names(datExpr)[!gsg$goodGenes], collapse = ", ")));
if (sum(!gsg$goodSamples) > 0)
printFlush(paste(
"Removing samples:",
paste(rownames(datExpr)[!gsg$goodSamples], collapse =", ")))
datExpr = datExpr[gsg$goodSamples, gsg$goodGenes]
}
## Removing samples: CSM5MCVB_P, CSM79HNY_P, ESM5GEYY_P, ESM718U9_P, MSM6J2N6_P, MSM9VZLX_PAfter removing the outliers, we need to choose a suitable soft threshold parameter for creating the modules as part of the WGCNA algorithm. This power value must produce a graph similar to a scale-free network. We can use the mean connectivity graphic for the selection of this power parameter.
# Choose a set of soft threshold parameters
powers = c(c(1:20), seq(from = 22, to=30, by=2))
sft = pickSoftThreshold(
datExpr, powerVector = powers, verbose = 5, dataIsExpr = TRUE,
RsquaredCut = 0.30)
## pickSoftThreshold: will use block size 127.
## pickSoftThreshold: calculating connectivity for given powers...
## ..working on genes 1 through 127 of 127
## Power SFT.R.sq slope truncated.R.sq mean.k. median.k. max.k.
## 1 1 0.1790 -0.972 0.85700 15.00000 1.48e+01 23.0000
## 2 2 0.3450 -0.729 0.63500 2.95000 2.85e+00 5.6900
## 3 3 0.2400 -4.130 0.12700 0.80400 7.36e-01 2.6100
## 4 4 0.3100 -4.870 0.19900 0.29200 2.14e-01 1.6700
## 5 5 0.2150 -4.030 -0.00880 0.13700 7.02e-02 1.2200
## 6 6 0.1800 -2.930 -0.05130 0.07910 2.46e-02 0.9570
## 7 7 0.1380 -2.240 -0.10100 0.05320 9.33e-03 0.7900
## 8 8 0.2200 -2.790 -0.00259 0.03940 3.71e-03 0.6690
## 9 9 0.0872 -1.450 -0.09130 0.03100 1.50e-03 0.5760
## 10 10 0.1190 -1.630 -0.07660 0.02540 6.73e-04 0.5020
## 11 11 0.0562 -1.250 -0.03470 0.02130 2.66e-04 0.4410
## 12 12 0.1620 -2.150 -0.07720 0.01810 1.12e-04 0.3910
## 13 13 0.2120 -2.350 -0.00949 0.01560 4.84e-05 0.3480
## 14 14 0.2480 -2.410 0.04950 0.01360 2.12e-05 0.3110
## 15 15 0.2890 -2.470 0.15200 0.01190 9.53e-06 0.2810
## 16 16 0.3030 -2.420 0.18400 0.01050 4.33e-06 0.2580
## 17 17 0.2410 -2.380 0.02450 0.00933 1.98e-06 0.2380
## 18 18 0.3190 -2.640 0.15400 0.00830 9.03e-07 0.2180
## 19 19 0.3320 -2.600 0.17700 0.00741 4.03e-07 0.2010
## 20 20 0.2370 -2.050 0.02180 0.00664 1.70e-07 0.1840
## 21 22 0.2350 -2.000 0.07520 0.00536 3.40e-08 0.1560
## 22 24 0.2430 -2.230 0.09560 0.00437 6.98e-09 0.1310
## 23 26 0.2370 -2.120 0.26200 0.00358 1.44e-09 0.1110
## 24 28 0.2490 -2.080 0.26200 0.00294 2.93e-10 0.0937
## 25 30 0.2490 -2.290 0.30100 0.00243 5.67e-11 0.0791In this step, we will conduct a one-step module detection based on the selected soft threshold parameter selected above.
power = sft$powerEstimate
net = blockwiseModules(
datExpr,
power = power,
corFnc="bicor",
corOptions=list(maxPOutliers=0.1),
networkType ="unsigned",
maxBlockSize = ncol(datExpr),
minModuleSize = 3,
TOMType = "unsigned",
reassignThreshold = 0,
mergeCutHeight = 0,
verbose = 3)
## Calculating module eigengenes block-wise from all genes
## Flagging genes and samples with too many missing values...
## ..step 1
## ..Working on block 1 .
## TOM calculation: adjacency..
## ..will not use multithreading.
## Fraction of slow calculations: 0.000000
## ..connectivity..
## ..matrix multiplication (system BLAS)..
## ..normalization..
## ..done.
## ....clustering..
## ....detecting modules..
## ....calculating module eigengenes..
## ....checking kME in modules..
## ..removing 3 genes from module 1 because their KME is too low.
## ..removing 3 genes from module 2 because their KME is too low.
## ..removing 2 genes from module 3 because their KME is too low.
## ..removing 1 genes from module 5 because their KME is too low.
## ..removing 1 genes from module 7 because their KME is too low.
## ..merging modules that are too close..
## mergeCloseModules: Merging modules whose distance is less than 0
## Calculating new MEs...
####################
# How many modules #
####################
ncol(net$MEs)
## [1] 14
table(net$colors)
##
## black blue brown green greenyellow grey
## 7 14 13 12 5 10
## magenta pink purple red salmon tan
## 6 6 5 11 3 5
## turquoise yellow
## 18 12The WGCNA algorithm produced 14 modules which we can visualize as follows.
##########################
# Plot module dendrogram #
##########################
eigenGenes <- net$MEs
MEDiss = 1-cor(eigenGenes)
METree = hclust(as.dist(MEDiss), method = "average")
plot(METree, main = "Clustering of module eigengenes", xlab = "", sub = "")
Next, we calculate hub genes for the modules and create the mapping files to proceed with the MSEA.
###########################################
# Re-calculate modules and find hub genes #
###########################################
moduleColors <- net$colors
MEs0 = moduleEigengenes(datExpr, moduleColors)$eigengenes
modules_data = orderMEs(MEs0)
#######################
# Create mapping file #
#######################
library(tidyverse)
feature_by_modules <- as.data.frame(net$colors)
feature_by_modules <- rownames_to_column(feature_by_modules)
colnames(feature_by_modules) <- c('Feature', 'Module')
features_mapping <- feature_by_modules
features_mapping$Module <- paste('ME', features_mapping$Module, sep = '')Finally, we will run the MSEA analysis on the modules we constructed using WGCNA. Here, we first create a wrapper for the MSEA analysis using the gsEasy package.
library(reshape2)
library(gsEasy)
################
# MSEA Wrapper #
################
run_MSEA <- function(
microbeSet, # A list
ranked_features, # Ranked list of featured
filter.count = 3,
seed = 1234,
fdr.correction = 'BH') {
###################
# Filter out sets #
##################
microbeSet0 <- microbeSet
cond <- sapply(microbeSet0, function(x) length(x) > filter.count)
microbeSet <- microbeSet0[cond]
lengthmicrobeSet <- as.data.frame(
reshape2::melt(lapply(microbeSet, function(x) length(x))))
colnames(lengthmicrobeSet) <- c('Freq','Set')
################
# Classic MSEA #
################
set.seed(seed)
enrichment <- as.data.frame(
sapply(microbeSet, function(set) gset(S = set, r = ranked_features)))
colnames(enrichment)<-'ES'
enrichment <- rownames_to_column(enrichment, 'Set')
enrichment <- merge(enrichment, lengthmicrobeSet, 'Set')
enrichment$qval <- p.adjust(enrichment$ES, fdr.correction)
##########
# Return #
##########
return(enrichment)
}Before running the MSEA, we also need to rank the differential analysis results from MaAsLin2. We use the topGo package to create a list of microbe sets from the mapping file created above.
###################
# Rank DA results #
###################
results <- fit_data$results |> filter(metadata == 'disease')
results$qval <- p.adjust(results$pval, 'BH')
results <- results[order(results$qval, decreasing = FALSE), ]
###################
# MSEA Processing #
###################
library(topGO)
module_map <- features_mapping
mod.gs <- tapply(module_map$Module, module_map$Feature, as.character)
microbeSet <- inverseList(mod.gs)
microbeSet
## $MEblack
## [1] "Anaeromassilibacillus.sp..An250" "Anaerotruncus.colihominis"
## [3] "Blautia.wexlerae" "Eisenbergiella.tayi"
## [5] "Firmicutes.bacterium.CAG.94" "Ruthenibacterium.lactatiformans"
## [7] "Sellimonas.intestinalis"
##
## $MEblue
## [1] "Clostridium.sp..CAG.58" "Erysipelatoclostridium.ramosum"
## [3] "Haemophilus.parainfluenzae" "Intestinibacter.bartlettii"
## [5] "Klebsiella.pneumoniae" "Klebsiella.variicola"
## [7] "Lawsonibacter.asaccharolyticus" "Streptococcus.parasanguinis"
## [9] "Streptococcus.salivarius" "Veillonella.atypica"
## [11] "Veillonella.dispar" "Veillonella.infantium"
## [13] "Veillonella.parvula" "Veillonella.sp..T11011.6"
##
## $MEbrown
## [1] "Alistipes.finegoldii" "Alistipes.indistinctus"
## [3] "Alistipes.putredinis" "Alistipes.shahii"
## [5] "Bacteroides.xylanisolvens" "Bilophila.wadsworthia"
## [7] "Firmicutes.bacterium.CAG.83" "Odoribacter.splanchnicus"
## [9] "Oscillibacter.sp..57_20" "Oscillibacter.sp..CAG.241"
## [11] "Phocaeicola.dorei" "Ruminococcus.bromii"
## [13] "X.Eubacterium..siraeum"
##
## $MEgreen
## [1] "Blautia.sp..CAG.257" "Clostridium.bolteae.CAG.59"
## [3] "Eggerthella.lenta" "Eisenbergiella.massiliensis"
## [5] "Enterocloster.aldenensis" "Enterocloster.bolteae"
## [7] "Enterocloster.citroniae" "Enterocloster.clostridioformis"
## [9] "Enterocloster.lavalensis" "Flavonifractor.plautii"
## [11] "Lacrimispora.saccharolytica" "X.Clostridium..symbiosum"
##
## $MEgreenyellow
## [1] "Coprobacter.fastidiosus" "Escherichia.coli"
## [3] "Hungatella.hathewayi" "X.Clostridium..innocuum"
## [5] "X.Ruminococcus..gnavus"
##
## $MEgrey
## [1] "Anaerotignum.lactatifermentans" "Bacteroides.cellulosilyticus"
## [3] "Bacteroides.fragilis" "Collinsella.intestinalis"
## [5] "Eubacterium.sp..CAG.38" "Haemophilus.sp..HMSC71H05"
## [7] "Intestinimonas.butyriciproducens" "Lachnospira.eligens"
## [9] "Roseburia.intestinalis" "Roseburia.sp..CAG.471"
##
## $MEmagenta
## [1] "Bacteroides.finegoldii" "Paraprevotella.clara"
## [3] "Paraprevotella.xylaniphila" "Phocaeicola.coprocola"
## [5] "Phocaeicola.plebeius" "Prevotella.copri"
##
## $MEpink
## [1] "Bacteroides.galacturonicus" "Bifidobacterium.pseudocatenulatum"
## [3] "Eubacterium.sp..CAG.251" "Lachnospira.pectinoschiza"
## [5] "Lactobacillus.rogosae" "Phocaeicola.massiliensis"
##
## $MEpurple
## [1] "Barnesiella.intestinihominis" "Butyricimonas.synergistica"
## [3] "Butyricimonas.virosa" "Coprococcus.eutactus"
## [5] "Ruminococcus.bicirculans"
##
## $MEred
## [1] "Akkermansia.muciniphila" "Bacteroides.intestinalis"
## [3] "Clostridium.sp..CAG.299" "Dialister.invisus"
## [5] "Dielma.fastidiosa" "Holdemania.filiformis"
## [7] "Monoglobus.pectinilyticus" "Parasutterella.excrementihominis"
## [9] "Proteobacteria.bacterium.CAG.139" "Turicimonas.muris"
## [11] "X.Clostridium..leptum"
##
## $MEsalmon
## [1] "Bacteroides.faecis" "Bacteroides.faecis.CAG.32"
## [3] "Phascolarctobacterium.faecium"
##
## $MEtan
## [1] "Bacteroides.salyersiae" "Bifidobacterium.adolescentis"
## [3] "Bifidobacterium.bifidum" "Bifidobacterium.longum"
## [5] "Collinsella.aerofaciens"
##
## $MEturquoise
## [1] "Agathobaculum.butyriciproducens" "Anaerobutyricum.hallii"
## [3] "Anaerostipes.hadrus" "Bacteroides.eggerthii"
## [5] "Blautia.obeum" "Coprococcus.catus"
## [7] "Coprococcus.comes" "Dorea.formicigenerans"
## [9] "Dorea.longicatena" "Eubacterium.ramulus"
## [11] "Faecalibacterium.prausnitzii" "Fusicatenibacter.saccharivorans"
## [13] "Gemmiger.formicilis" "Roseburia.faecis"
## [15] "Roseburia.hominis" "Roseburia.inulinivorans"
## [17] "X.Eubacterium..rectale" "X.Ruminococcus..torques"
##
## $MEyellow
## [1] "Acidaminococcus.intestini" "Bacteroides.caccae"
## [3] "Bacteroides.ovatus" "Bacteroides.stercoris"
## [5] "Bacteroides.thetaiotaomicron" "Bacteroides.uniformis"
## [7] "Eubacterium.ventriosum" "Parabacteroides.distasonis"
## [9] "Parabacteroides.merdae" "Phocaeicola.vulgatus"
## [11] "Ruminococcaceae.bacterium.D16" "X.Ruminococcus..lactaris"We are now ready to run the MSEA analysis. We run \(100,000\) permutations to calculate the enrichment scores.
We can plot the enrichment scores to visualize the MSEA results.
p <- MSEA |>
arrange(-pval) |>
mutate(ID = factor(ID, levels = ID)) |>
ggplot(aes(y = -log10(pval), x = ID)) +
geom_bar(stat = "identity", fill = 'cornflowerblue') + theme_bw() +
coord_flip() +
ggtitle('Statistically significant modules associated with disease') +
xlab('') +
ylab('MSEA enrichment score')
print(p)
Based on the MSEA results, we obtain 13 enriched modules of microbial species. We can also examine the members of the top enriched modules.
A.3 Print the most significant modules for species relative abundance data
microbeSet[["MEpurple"]]
## [1] "Barnesiella.intestinihominis" "Butyricimonas.synergistica"
## [3] "Butyricimonas.virosa" "Coprococcus.eutactus"
## [5] "Ruminococcus.bicirculans"
microbeSet[["MEbrown"]]
## [1] "Alistipes.finegoldii" "Alistipes.indistinctus"
## [3] "Alistipes.putredinis" "Alistipes.shahii"
## [5] "Bacteroides.xylanisolvens" "Bilophila.wadsworthia"
## [7] "Firmicutes.bacterium.CAG.83" "Odoribacter.splanchnicus"
## [9] "Oscillibacter.sp..57_20" "Oscillibacter.sp..CAG.241"
## [11] "Phocaeicola.dorei" "Ruminococcus.bromii"
## [13] "X.Eubacterium..siraeum"A.4 Performing the MSEA analysis with pathway relative abundance data
Next, we repeat the MSEA with the pathway relative abundance data from the iHMP project and follow the same steps as before.
##########################
# Load HMP2 pathway data #
##########################
se_pathway <- sampleMetadata |>
filter(study_name == "HMP_2019_ibdmdb") |>
returnSamples("pathway_abundance", rownames = "short")
##########################
# Create sample metadata #
##########################
sample_metadata <- colData(se_pathway) |>
as.data.frame() |> filter(visit_number == 1) |>
dplyr::select("age", "disease", "antibiotics_current_use")
# Set reference
sample_metadata$disease <- as.factor(sample_metadata$disease)
sample_metadata$disease <- relevel(sample_metadata$disease, 'healthy')
###########################
# Create Pathway Features #
###########################
feature_pwys_t <- as.data.frame(assay(se_pathway))
feature_pwys_t <- rownames_to_column(feature_pwys_t, "ID")
feature_pwys_t <- feature_pwys_t |>
filter(!grepl("\\|", ID)) |>
filter(!ID %in% c('UNMAPPED', 'UNINTEGRATED')) |>
column_to_rownames('ID') |>
as.data.frame()
##############################
# Subset to baseline samples #
##############################
feature_pwys <- as.data.frame(t(feature_pwys_t))
feature_pwys <- feature_pwys[rownames(sample_metadata),]
feature_pwys <- feature_pwys / 100
rm(feature_pwys_t); rm(se_pathway)As before, we first run a MaAsLin2 analysis using default settings and construct the modules using residuals from the MaAsLin2 models.
fit_data = Maaslin2(
input_data = feature_pwys,
input_metadata = sample_metadata,
normalization = "NONE",
output = "output_pwys",
fixed_effects = c("disease", "age", "antibiotics_current_use"))
## [1] "Creating output folder"
## [1] "Creating output feature tables folder"
## [1] "Creating output fits folder"
## [1] "Creating output figures folder"
## 2024-11-14 13:24:57.678246 INFO::Writing function arguments to log file
## 2024-11-14 13:24:57.681932 INFO::Verifying options selected are valid
## 2024-11-14 13:24:57.682391 INFO::Determining format of input files
## 2024-11-14 13:24:57.682837 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-11-14 13:24:57.688023 INFO::Formula for fixed effects: expr ~ disease + age + antibiotics_current_use
## 2024-11-14 13:24:57.688553 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
## 2024-11-14 13:24:57.689133 INFO::Filter data based on min abundance and min prevalence
## 2024-11-14 13:24:57.68957 INFO::Total samples in data: 136
## 2024-11-14 13:24:57.689989 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
## 2024-11-14 13:24:57.695285 INFO::Total filtered features: 113
## 2024-11-14 13:24:57.695906 INFO::Filtered feature names from abundance and prevalence filtering: PWY.5044..purine.nucleotides.degradation.I..plants., PROPFERM.PWY..L.alanine.fermentation.to.propanoate.and.acetate, PWY.6596..adenosine.nucleotides.degradation.I, PWY.5004..superpathway.of.L.citrulline.metabolism, UDPNACETYLGALSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.II, PWY66.367..ketogenesis, PWY.5392..reductive.TCA.cycle.II, PWY.101..photosynthesis.light.reactions, PWY.7031..protein.N.glycosylation..bacterial., PWY.5265..peptidoglycan.biosynthesis.II..staphylococci., PWY.7268..NAD.NADP.NADH.NADPH.cytosolic.interconversion..yeast., PWY.7165..L.ascorbate.biosynthesis.VI..engineered.pathway., CATECHOL.ORTHO.CLEAVAGE.PWY..catechol.degradation.to..beta..ketoadipate, PWY.5417..catechol.degradation.III..ortho.cleavage.pathway., PWY.5431..aromatic.compounds.degradation.via..beta..ketoadipate, PWY3DJ.35471..L.ascorbate.biosynthesis.IV, PWY.6185..4.methylcatechol.degradation..ortho.cleavage., PWY.7245..superpathway.NAD.NADP...NADH.NADPH.interconversion..yeast., PWY.5181..toluene.degradation.III..aerobic...via.p.cresol., PWY.6562..norspermidine.biosynthesis, PWY.7431..aromatic.biogenic.amine.degradation..bacteria., PWY.6307..L.tryptophan.degradation.X..mammalian..via.tryptamine., PWY.6313..serotonin.degradation, URSIN.PWY..ureide.biosynthesis, LIPASYN.PWY..phospholipases, DHGLUCONATE.PYR.CAT.PWY..glucose.degradation..oxidative., DENITRIFICATION.PWY..nitrate.reduction.I..denitrification., PWY.6662..superpathway.of.quinolone.and.alkylquinolone.biosynthesis, PWY.6660..2.heptyl.3.hydroxy.4.1H..quinolone.biosynthesis, PWY.6182..superpathway.of.salicylate.degradation, P165.PWY..superpathway.of.purines.degradation.in.plants, PWY66.388..fatty.acid..alpha..oxidation.III, TYRFUMCAT.PWY..L.tyrosine.degradation.I, PWY.5101..L.isoleucine.biosynthesis.II, PWY.6167..flavin.biosynthesis.II..archaea., PWY.6748..nitrate.reduction.VII..denitrification., PWY.7039..phosphatidate.metabolism..as.a.signaling.molecule, VALDEG.PWY..L.valine.degradation.I, PWY.5198..factor.420.biosynthesis, PWY.6215..4.chlorobenzoate.degradation, AEROBACTINSYN.PWY..aerobactin.biosynthesis, P562.PWY..myo.inositol.degradation.I, PWY.7409..phospholipid.remodeling..phosphatidylethanolamine..yeast., PWY.4722..creatinine.degradation.II, PWY.3801..sucrose.degradation.II..sucrose.synthase., PWY.7345..superpathway.of.anaerobic.sucrose.degradation, P125.PWY..superpathway.of..R.R..butanediol.biosynthesis, PWY.5994..palmitate.biosynthesis.I..animals.and.fungi., PWY.4321..L.glutamate.degradation.IV, PWY.7528..L.methionine.salvage.cycle.I..bacteria.and.plants., PWY.4361..S.methyl.5.thio..alpha..D.ribose.1.phosphate.degradation, PWY.1541..superpathway.of.taurine.degradation, PWY.5654..2.amino.3.carboxymuconate.semialdehyde.degradation.to.2.oxopentenoate, PWY.6210..2.aminophenol.degradation, PWY.6071..superpathway.of.phenylethylamine.degradation, PWY0.321..phenylacetate.degradation.I..aerobic., PWY.7200..superpathway.of.pyrimidine.deoxyribonucleoside.salvage, PWY.2221..Entner.Doudoroff.pathway.III..semi.phosphorylative., PWY.6992..1.5.anhydrofructose.degradation, PWY.7294..xylose.degradation.IV, PWY.6145..superpathway.of.sialic.acids.and.CMP.sialic.acids.biosynthesis, PWY.5180..toluene.degradation.I..aerobic...via.o.cresol., PWY.5182..toluene.degradation.II..aerobic...via.4.methylcatechol., PWY.5415..catechol.degradation.I..meta.cleavage.pathway., PWY.6785..hydrogen.production.VIII, PWY.5509..adenosylcobalamin.biosynthesis.from.cobyrinate.a.c.diamide.I, PWY.6641..superpathway.of.sulfolactate.degradation, PWY.7527..L.methionine.salvage.cycle.III, PWY.6396..superpathway.of.2.3.butanediol.biosynthesis, PWY.6467..Kdo.transfer.to.lipid.IVA.III..Chlamydia., X7ALPHADEHYDROX.PWY..cholate.degradation..bacteria..anaerobic., PWY.7374..1.4.dihydroxy.6.naphthoate.biosynthesis.I, PWY.6906..chitin.derivatives.degradation, PWY.5514..UDP.N.acetyl.D.galactosamine.biosynthesis.II, PWY.7317..superpathway.of.dTDP.glucose.derived.O.antigen.building.blocks.biosynthesis, PWY.7218..photosynthetic.3.hydroxybutanoate.biosynthesis..engineered., PHOTOALL.PWY..oxygenic.photosynthesis, PWY.6344..L.ornithine.degradation.II..Stickland.reaction., P621.PWY..nylon.6.oligomer.degradation, PWY.5028..L.histidine.degradation.II, CRNFORCAT.PWY..creatinine.degradation.I, PWY.6269..adenosylcobalamin.salvage.from.cobinamide.II, PWY.7389..superpathway.of.anaerobic.energy.metabolism..invertebrates., PWY.7384..anaerobic.energy.metabolism..invertebrates..mitochondrial., PWY.6165..chorismate.biosynthesis.II..archaea., PWY5F9.12..biphenyl.degradation, PWY.5647..2.nitrobenzoate.degradation.I, PWY.6138..CMP.N.acetylneuraminate.biosynthesis.I..eukaryotes., PWY.5910..superpathway.of.geranylgeranyldiphosphate.biosynthesis.I..via.mevalonate., PWY.922..mevalonate.pathway.I, PWY.6435..4.hydroxybenzoate.biosynthesis.V, PWY3O.1109..superpathway.of.4.hydroxybenzoate.biosynthesis..yeast., PWY.5754..4.hydroxybenzoate.biosynthesis.I..eukaryotes., PWY1G.0..mycothiol.biosynthesis, PWY.1501..mandelate.degradation.I, PWY.6107..chlorosalicylate.degradation, PWY.5534..propylene.degradation, PWY.7118..chitin.degradation.to.ethanol, PWY.7290..Escherichia.coli.serotype.O86.O.antigen.biosynthesis, PWY.181..photorespiration, PWY.1622..formaldehyde.assimilation.I..serine.pathway., CODH.PWY..reductive.acetyl.coenzyme.A.pathway, PWY.6349..CDP.archaeol.biosynthesis, PWY.6174..mevalonate.pathway.II..archaea., PWY.7286..7..3.amino.3.carboxypropyl..wyosine.biosynthesis, METHANOGENESIS.PWY..methanogenesis.from.H2.and.CO2, PWY.7391..isoprene.biosynthesis.II..engineered., PWY.5055..nicotinate.degradation.III, PWY.7399..methylphosphonate.degradation.II, P241.PWY..coenzyme.B.biosynthesis, PWY.5178..toluene.degradation.IV..aerobic...via.catechol., PWY.5420..catechol.degradation.II..meta.cleavage.pathway., PWY.5419..catechol.degradation.to.2.oxopent.4.enoate.II
## 2024-11-14 13:24:57.702022 INFO::Total filtered features with variance filtering: 0
## 2024-11-14 13:24:57.702542 INFO::Filtered feature names from variance filtering:
## 2024-11-14 13:24:57.702973 INFO::Running selected normalization method: NONE
## 2024-11-14 13:24:57.703388 INFO::Applying z-score to standardize continuous metadata
## 2024-11-14 13:24:57.706669 INFO::Running selected transform method: LOG
## 2024-11-14 13:24:57.714376 INFO::Running selected analysis method: LM
## 2024-11-14 13:24:57.71492 INFO::Fitting model to feature number 1, PWY.1042..glycolysis.IV..plant.cytosol.
## 2024-11-14 13:24:57.717618 INFO::Fitting model to feature number 2, DTDPRHAMSYN.PWY..dTDP.L.rhamnose.biosynthesis.I
## 2024-11-14 13:24:57.719936 INFO::Fitting model to feature number 3, PWY.5695..urate.biosynthesis.inosine.5..phosphate.degradation
## 2024-11-14 13:24:57.722221 INFO::Fitting model to feature number 4, PWY.6936..seleno.amino.acid.biosynthesis
## 2024-11-14 13:24:57.72453 INFO::Fitting model to feature number 5, ILEUSYN.PWY..L.isoleucine.biosynthesis.I..from.threonine.
## 2024-11-14 13:24:57.726818 INFO::Fitting model to feature number 6, PWY.7111..pyruvate.fermentation.to.isobutanol..engineered.
## 2024-11-14 13:24:57.729121 INFO::Fitting model to feature number 7, VALSYN.PWY..L.valine.biosynthesis
## 2024-11-14 13:24:57.731383 INFO::Fitting model to feature number 8, PWY.6609..adenine.and.adenosine.salvage.III
## 2024-11-14 13:24:57.733687 INFO::Fitting model to feature number 9, PWY.6737..starch.degradation.V
## 2024-11-14 13:24:57.736003 INFO::Fitting model to feature number 10, PWY.7219..adenosine.ribonucleotides.de.novo.biosynthesis
## 2024-11-14 13:24:57.738254 INFO::Fitting model to feature number 11, PWY0.1586..peptidoglycan.maturation..meso.diaminopimelate.containing.
## 2024-11-14 13:24:57.740514 INFO::Fitting model to feature number 12, ASPASN.PWY..superpathway.of.L.aspartate.and.L.asparagine.biosynthesis
## 2024-11-14 13:24:57.742751 INFO::Fitting model to feature number 13, PWY.5973..cis.vaccenate.biosynthesis
## 2024-11-14 13:24:57.745097 INFO::Fitting model to feature number 14, PWY.7221..guanosine.ribonucleotides.de.novo.biosynthesis
## 2024-11-14 13:24:57.747488 INFO::Fitting model to feature number 15, PWY.5686..UMP.biosynthesis
## 2024-11-14 13:24:57.749747 INFO::Fitting model to feature number 16, X1CMET2.PWY..N10.formyl.tetrahydrofolate.biosynthesis
## 2024-11-14 13:24:57.752051 INFO::Fitting model to feature number 17, PWY.6122..5.aminoimidazole.ribonucleotide.biosynthesis.II
## 2024-11-14 13:24:57.754348 INFO::Fitting model to feature number 18, PWY.6277..superpathway.of.5.aminoimidazole.ribonucleotide.biosynthesis
## 2024-11-14 13:24:57.756644 INFO::Fitting model to feature number 19, PWY.6385..peptidoglycan.biosynthesis.III..mycobacteria.
## 2024-11-14 13:24:57.758906 INFO::Fitting model to feature number 20, PWY.3841..folate.transformations.II
## 2024-11-14 13:24:57.761182 INFO::Fitting model to feature number 21, RIBOSYN2.PWY..flavin.biosynthesis.I..bacteria.and.plants.
## 2024-11-14 13:24:57.763458 INFO::Fitting model to feature number 22, PWY.6387..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.I..meso.diaminopimelate.containing.
## 2024-11-14 13:24:57.765709 INFO::Fitting model to feature number 23, THISYNARA.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.III..eukaryotes.
## 2024-11-14 13:24:57.767983 INFO::Fitting model to feature number 24, PWY.7663..gondoate.biosynthesis..anaerobic.
## 2024-11-14 13:24:57.770283 INFO::Fitting model to feature number 25, PWY.6386..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.II..lysine.containing.
## 2024-11-14 13:24:57.772557 INFO::Fitting model to feature number 26, PWY.6700..queuosine.biosynthesis
## 2024-11-14 13:24:57.774834 INFO::Fitting model to feature number 27, PEPTIDOGLYCANSYN.PWY..peptidoglycan.biosynthesis.I..meso.diaminopimelate.containing.
## 2024-11-14 13:24:57.777109 INFO::Fitting model to feature number 28, TRNA.CHARGING.PWY..tRNA.charging
## 2024-11-14 13:24:57.779344 INFO::Fitting model to feature number 29, PWY.6121..5.aminoimidazole.ribonucleotide.biosynthesis.I
## 2024-11-14 13:24:57.781603 INFO::Fitting model to feature number 30, HISTSYN.PWY..L.histidine.biosynthesis
## 2024-11-14 13:24:57.783888 INFO::Fitting model to feature number 31, PWY.7229..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.I
## 2024-11-14 13:24:57.786119 INFO::Fitting model to feature number 32, PWY.7199..pyrimidine.deoxyribonucleosides.salvage
## 2024-11-14 13:24:57.78836 INFO::Fitting model to feature number 33, PANTO.PWY..phosphopantothenate.biosynthesis.I
## 2024-11-14 13:24:57.790574 INFO::Fitting model to feature number 34, PWY.2942..L.lysine.biosynthesis.III
## 2024-11-14 13:24:57.792846 INFO::Fitting model to feature number 35, PWY.7237..myo...chiro..and.scillo.inositol.degradation
## 2024-11-14 13:24:57.795105 INFO::Fitting model to feature number 36, PWY.6168..flavin.biosynthesis.III..fungi.
## 2024-11-14 13:24:57.797361 INFO::Fitting model to feature number 37, COA.PWY.1..coenzyme.A.biosynthesis.II..mammalian.
## 2024-11-14 13:24:57.799634 INFO::Fitting model to feature number 38, PWY.5667..CDP.diacylglycerol.biosynthesis.I
## 2024-11-14 13:24:57.80188 INFO::Fitting model to feature number 39, PWY0.1319..CDP.diacylglycerol.biosynthesis.II
## 2024-11-14 13:24:57.804153 INFO::Fitting model to feature number 40, PWY.5097..L.lysine.biosynthesis.VI
## 2024-11-14 13:24:57.80643 INFO::Fitting model to feature number 41, ANAGLYCOLYSIS.PWY..glycolysis.III..from.glucose.
## 2024-11-14 13:24:57.808735 INFO::Fitting model to feature number 42, PWY.6123..inosine.5..phosphate.biosynthesis.I
## 2024-11-14 13:24:57.810975 INFO::Fitting model to feature number 43, ARGININE.SYN4.PWY..L.ornithine.de.novo..biosynthesis
## 2024-11-14 13:24:57.813302 INFO::Fitting model to feature number 44, PWY.6163..chorismate.biosynthesis.from.3.dehydroquinate
## 2024-11-14 13:24:57.815605 INFO::Fitting model to feature number 45, THRESYN.PWY..superpathway.of.L.threonine.biosynthesis
## 2024-11-14 13:24:57.817881 INFO::Fitting model to feature number 46, PYRIDNUCSYN.PWY..NAD.biosynthesis.I..from.aspartate.
## 2024-11-14 13:24:57.820142 INFO::Fitting model to feature number 47, PWY.6124..inosine.5..phosphate.biosynthesis.II
## 2024-11-14 13:24:57.822373 INFO::Fitting model to feature number 48, PWY.6147..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.I
## 2024-11-14 13:24:57.824661 INFO::Fitting model to feature number 49, PWY.7539..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.III..Chlamydia.
## 2024-11-14 13:24:57.826908 INFO::Fitting model to feature number 50, PWY.6151..S.adenosyl.L.methionine.cycle.I
## 2024-11-14 13:24:57.829176 INFO::Fitting model to feature number 51, SER.GLYSYN.PWY..superpathway.of.L.serine.and.glycine.biosynthesis.I
## 2024-11-14 13:24:57.831523 INFO::Fitting model to feature number 52, PWY.6126..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.II
## 2024-11-14 13:24:57.833778 INFO::Fitting model to feature number 53, PANTOSYN.PWY..pantothenate.and.coenzyme.A.biosynthesis.I
## 2024-11-14 13:24:57.836032 INFO::Fitting model to feature number 54, PWY.7228..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.I
## 2024-11-14 13:24:57.838259 INFO::Fitting model to feature number 55, COA.PWY..coenzyme.A.biosynthesis.I
## 2024-11-14 13:24:57.840542 INFO::Fitting model to feature number 56, PWY.4242..pantothenate.and.coenzyme.A.biosynthesis.III
## 2024-11-14 13:24:57.842785 INFO::Fitting model to feature number 57, PWY.6703..preQ0.biosynthesis
## 2024-11-14 13:24:57.845061 INFO::Fitting model to feature number 58, THISYN.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.I
## 2024-11-14 13:24:57.847313 INFO::Fitting model to feature number 59, HISDEG.PWY..L.histidine.degradation.I
## 2024-11-14 13:24:57.849618 INFO::Fitting model to feature number 60, PWY.5659..GDP.mannose.biosynthesis
## 2024-11-14 13:24:57.851881 INFO::Fitting model to feature number 61, PWY.5030..L.histidine.degradation.III
## 2024-11-14 13:24:57.854212 INFO::Fitting model to feature number 62, PWY.5484..glycolysis.II..from.fructose.6.phosphate.
## 2024-11-14 13:24:57.856481 INFO::Fitting model to feature number 63, PWY.3001..superpathway.of.L.isoleucine.biosynthesis.I
## 2024-11-14 13:24:57.85872 INFO::Fitting model to feature number 64, PWY.6125..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.II
## 2024-11-14 13:24:57.860987 INFO::Fitting model to feature number 65, TRPSYN.PWY..L.tryptophan.biosynthesis
## 2024-11-14 13:24:57.863234 INFO::Fitting model to feature number 66, GLYCOLYSIS..glycolysis.I..from.glucose.6.phosphate.
## 2024-11-14 13:24:57.865514 INFO::Fitting model to feature number 67, PWY.724..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.II
## 2024-11-14 13:24:57.867776 INFO::Fitting model to feature number 68, PWY.6897..thiamin.salvage.II
## 2024-11-14 13:24:57.870047 INFO::Fitting model to feature number 69, BRANCHED.CHAIN.AA.SYN.PWY..superpathway.of.branched.amino.acid.biosynthesis
## 2024-11-14 13:24:57.872304 INFO::Fitting model to feature number 70, CALVIN.PWY..Calvin.Benson.Bassham.cycle
## 2024-11-14 13:24:57.87455 INFO::Fitting model to feature number 71, PWY.841..superpathway.of.purine.nucleotides.de.novo.biosynthesis.I
## 2024-11-14 13:24:57.876795 INFO::Fitting model to feature number 72, COMPLETE.ARO.PWY..superpathway.of.aromatic.amino.acid.biosynthesis
## 2024-11-14 13:24:57.879048 INFO::Fitting model to feature number 73, PWY.7220..adenosine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2024-11-14 13:24:57.881354 INFO::Fitting model to feature number 74, PWY.7222..guanosine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2024-11-14 13:24:57.883632 INFO::Fitting model to feature number 75, DENOVOPURINE2.PWY..superpathway.of.purine.nucleotides.de.novo.biosynthesis.II
## 2024-11-14 13:24:57.885913 INFO::Fitting model to feature number 76, PWY.7357..thiamin.formation.from.pyrithiamine.and.oxythiamine..yeast.
## 2024-11-14 13:24:57.888175 INFO::Fitting model to feature number 77, PWY.5103..L.isoleucine.biosynthesis.III
## 2024-11-14 13:24:57.890426 INFO::Fitting model to feature number 78, ARO.PWY..chorismate.biosynthesis.I
## 2024-11-14 13:24:57.892687 INFO::Fitting model to feature number 79, PWY.7282..4.amino.2.methyl.5.phosphomethylpyrimidine.biosynthesis..yeast.
## 2024-11-14 13:24:57.894987 INFO::Fitting model to feature number 80, PWY0.845..superpathway.of.pyridoxal.5..phosphate.biosynthesis.and.salvage
## 2024-11-14 13:24:57.897968 INFO::Fitting model to feature number 81, ANAEROFRUCAT.PWY..homolactic.fermentation
## 2024-11-14 13:24:57.902005 INFO::Fitting model to feature number 82, PWY.6892..thiazole.biosynthesis.I..E..coli.
## 2024-11-14 13:24:57.906049 INFO::Fitting model to feature number 83, RHAMCAT.PWY..L.rhamnose.degradation.I
## 2024-11-14 13:24:57.908939 INFO::Fitting model to feature number 84, PYRIDOXSYN.PWY..pyridoxal.5..phosphate.biosynthesis.I
## 2024-11-14 13:24:57.911199 INFO::Fitting model to feature number 85, PWY.1269..CMP.3.deoxy.D.manno.octulosonate.biosynthesis.I
## 2024-11-14 13:24:57.913516 INFO::Fitting model to feature number 86, PHOSLIPSYN.PWY..superpathway.of.phospholipid.biosynthesis.I..bacteria.
## 2024-11-14 13:24:57.915787 INFO::Fitting model to feature number 87, CITRULBIO.PWY..L.citrulline.biosynthesis
## 2024-11-14 13:24:57.918013 INFO::Fitting model to feature number 88, NAGLIPASYN.PWY..lipid.IVA.biosynthesis
## 2024-11-14 13:24:57.92027 INFO::Fitting model to feature number 89, BIOTIN.BIOSYNTHESIS.PWY..biotin.biosynthesis.I
## 2024-11-14 13:24:57.922507 INFO::Fitting model to feature number 90, PWY.5154..L.arginine.biosynthesis.III..via.N.acetyl.L.citrulline.
## 2024-11-14 13:24:57.924772 INFO::Fitting model to feature number 91, FASYN.ELONG.PWY..fatty.acid.elongation....saturated
## 2024-11-14 13:24:57.927042 INFO::Fitting model to feature number 92, PENTOSE.P.PWY..pentose.phosphate.pathway
## 2024-11-14 13:24:57.92933 INFO::Fitting model to feature number 93, PWYG.321..mycolate.biosynthesis
## 2024-11-14 13:24:57.93163 INFO::Fitting model to feature number 94, PWY.6519..8.amino.7.oxononanoate.biosynthesis.I
## 2024-11-14 13:24:57.933875 INFO::Fitting model to feature number 95, PWY.7664..oleate.biosynthesis.IV..anaerobic.
## 2024-11-14 13:24:57.936155 INFO::Fitting model to feature number 96, PWY.4981..L.proline.biosynthesis.II..from.arginine.
## 2024-11-14 13:24:57.938412 INFO::Fitting model to feature number 97, GLYCOGENSYNTH.PWY..glycogen.biosynthesis.I..from.ADP.D.Glucose.
## 2024-11-14 13:24:57.940662 INFO::Fitting model to feature number 98, PWY.7388..octanoyl..acyl.carrier.protein..biosynthesis..mitochondria..yeast.
## 2024-11-14 13:24:57.942929 INFO::Fitting model to feature number 99, NONOXIPENT.PWY..pentose.phosphate.pathway..non.oxidative.branch.
## 2024-11-14 13:24:57.9452 INFO::Fitting model to feature number 100, COBALSYN.PWY..adenosylcobalamin.salvage.from.cobinamide.I
## 2024-11-14 13:24:57.947478 INFO::Fitting model to feature number 101, PWY.5989..stearate.biosynthesis.II..bacteria.and.plants.
## 2024-11-14 13:24:57.949723 INFO::Fitting model to feature number 102, PWY.6282..palmitoleate.biosynthesis.I..from..5Z..dodec.5.enoate.
## 2024-11-14 13:24:57.951979 INFO::Fitting model to feature number 103, PWY0.862...5Z..dodec.5.enoate.biosynthesis
## 2024-11-14 13:24:57.954235 INFO::Fitting model to feature number 104, PWY.7400..L.arginine.biosynthesis.IV..archaebacteria.
## 2024-11-14 13:24:57.95655 INFO::Fitting model to feature number 105, ARGSYN.PWY..L.arginine.biosynthesis.I..via.L.ornithine.
## 2024-11-14 13:24:57.958809 INFO::Fitting model to feature number 106, PWY.4984..urea.cycle
## 2024-11-14 13:24:57.961069 INFO::Fitting model to feature number 107, FASYN.INITIAL.PWY..superpathway.of.fatty.acid.biosynthesis.initiation..E..coli.
## 2024-11-14 13:24:57.963316 INFO::Fitting model to feature number 108, PWY4FS.7..phosphatidylglycerol.biosynthesis.I..plastidic.
## 2024-11-14 13:24:57.965592 INFO::Fitting model to feature number 109, PWY4FS.8..phosphatidylglycerol.biosynthesis.II..non.plastidic.
## 2024-11-14 13:24:57.967865 INFO::Fitting model to feature number 110, ARGSYNBSUB.PWY..L.arginine.biosynthesis.II..acetyl.cycle.
## 2024-11-14 13:24:57.970111 INFO::Fitting model to feature number 111, PWY.6317..galactose.degradation.I..Leloir.pathway.
## 2024-11-14 13:24:57.972359 INFO::Fitting model to feature number 112, PWY66.422..D.galactose.degradation.V..Leloir.pathway.
## 2024-11-14 13:24:57.974574 INFO::Fitting model to feature number 113, GLUTORN.PWY..L.ornithine.biosynthesis
## 2024-11-14 13:24:57.97684 INFO::Fitting model to feature number 114, PWY.2941..L.lysine.biosynthesis.II
## 2024-11-14 13:24:57.979092 INFO::Fitting model to feature number 115, PWY0.1296..purine.ribonucleosides.degradation
## 2024-11-14 13:24:57.981348 INFO::Fitting model to feature number 116, NAD.BIOSYNTHESIS.II..NAD.salvage.pathway.II
## 2024-11-14 13:24:57.983601 INFO::Fitting model to feature number 117, HSERMETANA.PWY..L.methionine.biosynthesis.III
## 2024-11-14 13:24:57.98585 INFO::Fitting model to feature number 118, PWY0.162..superpathway.of.pyrimidine.ribonucleotides.de.novo.biosynthesis
## 2024-11-14 13:24:57.988098 INFO::Fitting model to feature number 119, PWY.7208..superpathway.of.pyrimidine.nucleobases.salvage
## 2024-11-14 13:24:57.990305 INFO::Fitting model to feature number 120, TEICHOICACID.PWY..teichoic.acid..poly.glycerol..biosynthesis
## 2024-11-14 13:24:57.992557 INFO::Fitting model to feature number 121, PWY.6305..putrescine.biosynthesis.IV
## 2024-11-14 13:24:57.994909 INFO::Fitting model to feature number 122, GLUCONEO.PWY..gluconeogenesis.I
## 2024-11-14 13:24:57.997198 INFO::Fitting model to feature number 123, PWY.621..sucrose.degradation.III..sucrose.invertase.
## 2024-11-14 13:24:57.999503 INFO::Fitting model to feature number 124, PWY.6901..superpathway.of.glucose.and.xylose.degradation
## 2024-11-14 13:24:58.001763 INFO::Fitting model to feature number 125, POLYISOPRENSYN.PWY..polyisoprenoid.biosynthesis..E..coli.
## 2024-11-14 13:24:58.004043 INFO::Fitting model to feature number 126, PWY.7323..superpathway.of.GDP.mannose.derived.O.antigen.building.blocks.biosynthesis
## 2024-11-14 13:24:58.006289 INFO::Fitting model to feature number 127, PWY.5941..glycogen.degradation.II..eukaryotic.
## 2024-11-14 13:24:58.008583 INFO::Fitting model to feature number 128, PWY.6527..stachyose.degradation
## 2024-11-14 13:24:58.010918 INFO::Fitting model to feature number 129, PWY0.1261..anhydromuropeptides.recycling
## 2024-11-14 13:24:58.013187 INFO::Fitting model to feature number 130, PWY66.400..glycolysis.VI..metazoan.
## 2024-11-14 13:24:58.015472 INFO::Fitting model to feature number 131, PWY0.166..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis..E..coli.
## 2024-11-14 13:24:58.017792 INFO::Fitting model to feature number 132, GLYCOLYSIS.E.D..superpathway.of.glycolysis.and.Entner.Doudoroff
## 2024-11-14 13:24:58.020048 INFO::Fitting model to feature number 133, PWY.7187..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2024-11-14 13:24:58.02225 INFO::Fitting model to feature number 134, PWY.7456..mannan.degradation
## 2024-11-14 13:24:58.024522 INFO::Fitting model to feature number 135, PWY.7184..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.I
## 2024-11-14 13:24:58.026754 INFO::Fitting model to feature number 136, OANTIGEN.PWY..O.antigen.building.blocks.biosynthesis..E..coli.
## 2024-11-14 13:24:58.028997 INFO::Fitting model to feature number 137, COLANSYN.PWY..colanic.acid.building.blocks.biosynthesis
## 2024-11-14 13:24:58.031225 INFO::Fitting model to feature number 138, PWY.6545..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.III
## 2024-11-14 13:24:58.033523 INFO::Fitting model to feature number 139, PWY.6895..superpathway.of.thiamin.diphosphate.biosynthesis.II
## 2024-11-14 13:24:58.035801 INFO::Fitting model to feature number 140, PWY.6608..guanosine.nucleotides.degradation.III
## 2024-11-14 13:24:58.038083 INFO::Fitting model to feature number 141, TCA..TCA.cycle.I..prokaryotic.
## 2024-11-14 13:24:58.040334 INFO::Fitting model to feature number 142, PWY.6859..all.trans.farnesol.biosynthesis
## 2024-11-14 13:24:58.042553 INFO::Fitting model to feature number 143, PWY.5100..pyruvate.fermentation.to.acetate.and.lactate.II
## 2024-11-14 13:24:58.044834 INFO::Fitting model to feature number 144, PWY.6572..chondroitin.sulfate.degradation.I..bacterial.
## 2024-11-14 13:24:58.047055 INFO::Fitting model to feature number 145, PWY.6471..peptidoglycan.biosynthesis.IV..Enterococcus.faecium.
## 2024-11-14 13:24:58.049299 INFO::Fitting model to feature number 146, PWY.6470..peptidoglycan.biosynthesis.V...beta..lactam.resistance.
## 2024-11-14 13:24:58.051559 INFO::Fitting model to feature number 147, PWY.5188..tetrapyrrole.biosynthesis.I..from.glutamate.
## 2024-11-14 13:24:58.05379 INFO::Fitting model to feature number 148, GLUCUROCAT.PWY..superpathway.of..beta..D.glucuronide.and.D.glucuronate.degradation
## 2024-11-14 13:24:58.05604 INFO::Fitting model to feature number 149, PWY.6749..CMP.legionaminate.biosynthesis.I
## 2024-11-14 13:24:58.058283 INFO::Fitting model to feature number 150, P164.PWY..purine.nucleobases.degradation.I..anaerobic.
## 2024-11-14 13:24:58.060527 INFO::Fitting model to feature number 151, PWY.7198..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.IV
## 2024-11-14 13:24:58.062753 INFO::Fitting model to feature number 152, PWY.7211..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis
## 2024-11-14 13:24:58.064993 INFO::Fitting model to feature number 153, PWY66.409..superpathway.of.purine.nucleotide.salvage
## 2024-11-14 13:24:58.06724 INFO::Fitting model to feature number 154, GALACT.GLUCUROCAT.PWY..superpathway.of.hexuronide.and.hexuronate.degradation
## 2024-11-14 13:24:58.06956 INFO::Fitting model to feature number 155, UDPNAGSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.I
## 2024-11-14 13:24:58.071848 INFO::Fitting model to feature number 156, PWY.6353..purine.nucleotides.degradation.II..aerobic.
## 2024-11-14 13:24:58.074131 INFO::Fitting model to feature number 157, PWY.7197..pyrimidine.deoxyribonucleotide.phosphorylation
## 2024-11-14 13:24:58.076395 INFO::Fitting model to feature number 158, PWY.7242..D.fructuronate.degradation
## 2024-11-14 13:24:58.078645 INFO::Fitting model to feature number 159, PWY.7196..superpathway.of.pyrimidine.ribonucleosides.salvage
## 2024-11-14 13:24:58.080937 INFO::Fitting model to feature number 160, PWY.5690..TCA.cycle.II..plants.and.fungi.
## 2024-11-14 13:24:58.083233 INFO::Fitting model to feature number 161, PWY.5177..glutaryl.CoA.degradation
## 2024-11-14 13:24:58.085499 INFO::Fitting model to feature number 162, PWY.6478..GDP.D.glycero..alpha..D.manno.heptose.biosynthesis
## 2024-11-14 13:24:58.08778 INFO::Fitting model to feature number 163, HEXITOLDEGSUPER.PWY..superpathway.of.hexitol.degradation..bacteria.
## 2024-11-14 13:24:58.090017 INFO::Fitting model to feature number 164, GALACTUROCAT.PWY..D.galacturonate.degradation.I
## 2024-11-14 13:24:58.092294 INFO::Fitting model to feature number 165, PWY.5104..L.isoleucine.biosynthesis.IV
## 2024-11-14 13:24:58.094548 INFO::Fitting model to feature number 166, PWY.5505..L.glutamate.and.L.glutamine.biosynthesis
## 2024-11-14 13:24:58.096805 INFO::Fitting model to feature number 167, P441.PWY..superpathway.of.N.acetylneuraminate.degradation
## 2024-11-14 13:24:58.099096 INFO::Fitting model to feature number 168, PWY.6606..guanosine.nucleotides.degradation.II
## 2024-11-14 13:24:58.101371 INFO::Fitting model to feature number 169, SALVADEHYPOX.PWY..adenosine.nucleotides.degradation.II
## 2024-11-14 13:24:58.103662 INFO::Fitting model to feature number 170, PWY.5005..biotin.biosynthesis.II
## 2024-11-14 13:24:58.105934 INFO::Fitting model to feature number 171, PWY.6549..L.glutamine.biosynthesis.III
## 2024-11-14 13:24:58.108211 INFO::Fitting model to feature number 172, GLCMANNANAUT.PWY..superpathway.of.N.acetylglucosamine..N.acetylmannosamine.and.N.acetylneuraminate.degradation
## 2024-11-14 13:24:58.110451 INFO::Fitting model to feature number 173, PWY0.1241..ADP.L.glycero..beta..D.manno.heptose.biosynthesis
## 2024-11-14 13:24:58.112755 INFO::Fitting model to feature number 174, PWY.6507..4.deoxy.L.threo.hex.4.enopyranuronate.degradation
## 2024-11-14 13:24:58.11506 INFO::Fitting model to feature number 175, GALACTARDEG.PWY..D.galactarate.degradation.I
## 2024-11-14 13:24:58.117393 INFO::Fitting model to feature number 176, GLUCARGALACTSUPER.PWY..superpathway.of.D.glucarate.and.D.galactarate.degradation
## 2024-11-14 13:24:58.119681 INFO::Fitting model to feature number 177, PWY.5676..acetyl.CoA.fermentation.to.butanoate.II
## 2024-11-14 13:24:58.121915 INFO::Fitting model to feature number 178, PWY.5347..superpathway.of.L.methionine.biosynthesis..transsulfuration.
## 2024-11-14 13:24:58.12424 INFO::Fitting model to feature number 179, PWY.5138..unsaturated..even.numbered.fatty.acid..beta..oxidation
## 2024-11-14 13:24:58.126491 INFO::Fitting model to feature number 180, MET.SAM.PWY..superpathway.of.S.adenosyl.L.methionine.biosynthesis
## 2024-11-14 13:24:58.128792 INFO::Fitting model to feature number 181, PWY66.399..gluconeogenesis.III
## 2024-11-14 13:24:58.131102 INFO::Fitting model to feature number 182, PWY0.1297..superpathway.of.purine.deoxyribonucleosides.degradation
## 2024-11-14 13:24:58.133415 INFO::Fitting model to feature number 183, METSYN.PWY..L.homoserine.and.L.methionine.biosynthesis
## 2024-11-14 13:24:58.135814 INFO::Fitting model to feature number 184, PWY.7332..superpathway.of.UDP.N.acetylglucosamine.derived.O.antigen.building.blocks.biosynthesis
## 2024-11-14 13:24:58.138108 INFO::Fitting model to feature number 185, PWY.6588..pyruvate.fermentation.to.acetone
## 2024-11-14 13:24:58.140429 INFO::Fitting model to feature number 186, PWY.6891..thiazole.biosynthesis.II..Bacillus.
## 2024-11-14 13:24:58.14273 INFO::Fitting model to feature number 187, GLUCARDEG.PWY..D.glucarate.degradation.I
## 2024-11-14 13:24:58.145059 INFO::Fitting model to feature number 188, METH.ACETATE.PWY..methanogenesis.from.acetate
## 2024-11-14 13:24:58.147396 INFO::Fitting model to feature number 189, PWY.7013..L.1.2.propanediol.degradation
## 2024-11-14 13:24:58.149745 INFO::Fitting model to feature number 190, PWY.6590..superpathway.of.Clostridium.acetobutylicum.acidogenic.fermentation
## 2024-11-14 13:24:58.152046 INFO::Fitting model to feature number 191, FAO.PWY..fatty.acid..beta..oxidation.I
## 2024-11-14 13:24:58.154339 INFO::Fitting model to feature number 192, PWY.6318..L.phenylalanine.degradation.IV..mammalian..via.side.chain.
## 2024-11-14 13:24:58.156626 INFO::Fitting model to feature number 193, CENTFERM.PWY..pyruvate.fermentation.to.butanoate
## 2024-11-14 13:24:58.158938 INFO::Fitting model to feature number 194, GOLPDLCAT.PWY..superpathway.of.glycerol.degradation.to.1.3.propanediol
## 2024-11-14 13:24:58.161223 INFO::Fitting model to feature number 195, PWY.5136..fatty.acid..beta..oxidation.II..peroxisome.
## 2024-11-14 13:24:58.163506 INFO::Fitting model to feature number 196, PWY66.391..fatty.acid..beta..oxidation.VI..peroxisome.
## 2024-11-14 13:24:58.165852 INFO::Fitting model to feature number 197, HOMOSER.METSYN.PWY..L.methionine.biosynthesis.I
## 2024-11-14 13:24:58.16819 INFO::Fitting model to feature number 198, METHGLYUT.PWY..superpathway.of.methylglyoxal.degradation
## 2024-11-14 13:24:58.170484 INFO::Fitting model to feature number 199, PWY.7288..fatty.acid..beta..oxidation..peroxisome..yeast.
## 2024-11-14 13:24:58.172784 INFO::Fitting model to feature number 200, PWY.7383..anaerobic.energy.metabolism..invertebrates..cytosol.
## 2024-11-14 13:24:58.175058 INFO::Fitting model to feature number 201, P162.PWY..L.glutamate.degradation.V..via.hydroxyglutarate.
## 2024-11-14 13:24:58.177384 INFO::Fitting model to feature number 202, PWY.4041...gamma..glutamyl.cycle
## 2024-11-14 13:24:58.179718 INFO::Fitting model to feature number 203, P108.PWY..pyruvate.fermentation.to.propanoate.I
## 2024-11-14 13:24:58.181996 INFO::Fitting model to feature number 204, PWY.5022..4.aminobutanoate.degradation.V
## 2024-11-14 13:24:58.184284 INFO::Fitting model to feature number 205, GLUDEG.I.PWY..GABA.shunt
## 2024-11-14 13:24:58.186544 INFO::Fitting model to feature number 206, PWY.5677..succinate.fermentation.to.butanoate
## 2024-11-14 13:24:58.188858 INFO::Fitting model to feature number 207, P163.PWY..L.lysine.fermentation.to.acetate.and.butanoate
## 2024-11-14 13:24:58.191155 INFO::Fitting model to feature number 208, PWY.3781..aerobic.respiration.I..cytochrome.c.
## 2024-11-14 13:24:58.193438 INFO::Fitting model to feature number 209, PWY.5083..NAD.NADH.phosphorylation.and.dephosphorylation
## 2024-11-14 13:24:58.195779 INFO::Fitting model to feature number 210, DAPLYSINESYN.PWY..L.lysine.biosynthesis.I
## 2024-11-14 13:24:58.198062 INFO::Fitting model to feature number 211, P461.PWY..hexitol.fermentation.to.lactate..formate..ethanol.and.acetate
## 2024-11-14 13:24:58.20035 INFO::Fitting model to feature number 212, PWY.1861..formaldehyde.assimilation.II..RuMP.Cycle.
## 2024-11-14 13:24:58.202662 INFO::Fitting model to feature number 213, PWY.7312..dTDP.D..beta..fucofuranose.biosynthesis
## 2024-11-14 13:24:58.204975 INFO::Fitting model to feature number 214, PWY.6630..superpathway.of.L.tyrosine.biosynthesis
## 2024-11-14 13:24:58.207261 INFO::Fitting model to feature number 215, PWY.6595..superpathway.of.guanosine.nucleotides.degradation..plants.
## 2024-11-14 13:24:58.20961 INFO::Fitting model to feature number 216, ARG.POLYAMINE.SYN..superpathway.of.arginine.and.polyamine.biosynthesis
## 2024-11-14 13:24:58.2119 INFO::Fitting model to feature number 217, PWY.7003..glycerol.degradation.to.butanol
## 2024-11-14 13:24:58.214186 INFO::Fitting model to feature number 218, P185.PWY..formaldehyde.assimilation.III..dihydroxyacetone.cycle.
## 2024-11-14 13:24:58.216579 INFO::Fitting model to feature number 219, POLYAMSYN.PWY..superpathway.of.polyamine.biosynthesis.I
## 2024-11-14 13:24:58.21888 INFO::Fitting model to feature number 220, PWY0.1298..superpathway.of.pyrimidine.deoxyribonucleosides.degradation
## 2024-11-14 13:24:58.221215 INFO::Fitting model to feature number 221, LACTOSECAT.PWY..lactose.and.galactose.degradation.I
## 2024-11-14 13:24:58.22356 INFO::Fitting model to feature number 222, PWY4LZ.257..superpathway.of.fermentation..Chlamydomonas.reinhardtii.
## 2024-11-14 13:24:58.225857 INFO::Fitting model to feature number 223, P161.PWY..acetylene.degradation
## 2024-11-14 13:24:58.228168 INFO::Fitting model to feature number 224, RUMP.PWY..formaldehyde.oxidation.I
## 2024-11-14 13:24:58.230465 INFO::Fitting model to feature number 225, PPGPPMET.PWY..ppGpp.biosynthesis
## 2024-11-14 13:24:58.23283 INFO::Fitting model to feature number 226, PWY.5304..superpathway.of.sulfur.oxidation..Acidianus.ambivalens.
## 2024-11-14 13:24:58.235117 INFO::Fitting model to feature number 227, PWY.6531..mannitol.cycle
## 2024-11-14 13:24:58.237433 INFO::Fitting model to feature number 228, FUC.RHAMCAT.PWY..superpathway.of.fucose.and.rhamnose.degradation
## 2024-11-14 13:24:58.240108 INFO::Fitting model to feature number 229, PWY.7315..dTDP.N.acetylthomosamine.biosynthesis
## 2024-11-14 13:24:58.267233 INFO::Fitting model to feature number 230, FUCCAT.PWY..fucose.degradation
## 2024-11-14 13:24:58.269875 INFO::Fitting model to feature number 231, PWY.7210..pyrimidine.deoxyribonucleotides.biosynthesis.from.CTP
## 2024-11-14 13:24:58.272188 INFO::Fitting model to feature number 232, FOLSYN.PWY..superpathway.of.tetrahydrofolate.biosynthesis.and.salvage
## 2024-11-14 13:24:58.274443 INFO::Fitting model to feature number 233, PWY.6612..superpathway.of.tetrahydrofolate.biosynthesis
## 2024-11-14 13:24:58.276748 INFO::Fitting model to feature number 234, PWY0.1061..superpathway.of.L.alanine.biosynthesis
## 2024-11-14 13:24:58.278983 INFO::Fitting model to feature number 235, PWY.5189..tetrapyrrole.biosynthesis.II..from.glycine.
## 2024-11-14 13:24:58.28126 INFO::Fitting model to feature number 236, PWY.6876..isopropanol.biosynthesis
## 2024-11-14 13:24:58.283531 INFO::Fitting model to feature number 237, PWY0.781..aspartate.superpathway
## 2024-11-14 13:24:58.285796 INFO::Fitting model to feature number 238, P4.PWY..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.I
## 2024-11-14 13:24:58.288067 INFO::Fitting model to feature number 239, PWY0.1479..tRNA.processing
## 2024-11-14 13:24:58.290303 INFO::Fitting model to feature number 240, GLYCOLYSIS.TCA.GLYOX.BYPASS..superpathway.of.glycolysis..pyruvate.dehydrogenase..TCA..and.glyoxylate.bypass
## 2024-11-14 13:24:58.292561 INFO::Fitting model to feature number 241, PYRIDNUCSAL.PWY..NAD.salvage.pathway.I
## 2024-11-14 13:24:58.294812 INFO::Fitting model to feature number 242, GLYCOCAT.PWY..glycogen.degradation.I..bacterial.
## 2024-11-14 13:24:58.297084 INFO::Fitting model to feature number 243, PWY.5723..Rubisco.shunt
## 2024-11-14 13:24:58.299374 INFO::Fitting model to feature number 244, TCA.GLYOX.BYPASS..superpathway.of.glyoxylate.bypass.and.TCA
## 2024-11-14 13:24:58.301666 INFO::Fitting model to feature number 245, PWY.7385..1.3.propanediol.biosynthesis..engineered.
## 2024-11-14 13:24:58.303949 INFO::Fitting model to feature number 246, PWY.7254..TCA.cycle.VII..acetate.producers.
## 2024-11-14 13:24:58.306185 INFO::Fitting model to feature number 247, GLYOXYLATE.BYPASS..glyoxylate.cycle
## 2024-11-14 13:24:58.308463 INFO::Fitting model to feature number 248, PWY.6731..starch.degradation.III
## 2024-11-14 13:24:58.310706 INFO::Fitting model to feature number 249, PWY.7328..superpathway.of.UDP.glucose.derived.O.antigen.building.blocks.biosynthesis
## 2024-11-14 13:24:58.312968 INFO::Fitting model to feature number 250, POLYAMINSYN3.PWY..superpathway.of.polyamine.biosynthesis.II
## 2024-11-14 13:24:58.3152 INFO::Fitting model to feature number 251, PWY.2723..trehalose.degradation.V
## 2024-11-14 13:24:58.317614 INFO::Fitting model to feature number 252, GLUCOSE1PMETAB.PWY..glucose.and.glucose.1.phosphate.degradation
## 2024-11-14 13:24:58.319936 INFO::Fitting model to feature number 253, PWY.5384..sucrose.degradation.IV..sucrose.phosphorylase.
## 2024-11-14 13:24:58.322193 INFO::Fitting model to feature number 254, PWY.7046..4.coumarate.degradation..anaerobic.
## 2024-11-14 13:24:58.324625 INFO::Fitting model to feature number 255, PWY66.389..phytol.degradation
## 2024-11-14 13:24:58.326964 INFO::Fitting model to feature number 256, PWY.5971..palmitate.biosynthesis.II..bacteria.and.plants.
## 2024-11-14 13:24:58.329402 INFO::Fitting model to feature number 257, PWY.6629..superpathway.of.L.tryptophan.biosynthesis
## 2024-11-14 13:24:58.331701 INFO::Fitting model to feature number 258, PWY.5345..superpathway.of.L.methionine.biosynthesis..by.sulfhydrylation.
## 2024-11-14 13:24:58.333966 INFO::Fitting model to feature number 259, SULFATE.CYS.PWY..superpathway.of.sulfate.assimilation.and.cysteine.biosynthesis
## 2024-11-14 13:24:58.33626 INFO::Fitting model to feature number 260, PWY.5838..superpathway.of.menaquinol.8.biosynthesis.I
## 2024-11-14 13:24:58.338511 INFO::Fitting model to feature number 261, PRPP.PWY..superpathway.of.histidine..purine..and.pyrimidine.biosynthesis
## 2024-11-14 13:24:58.340821 INFO::Fitting model to feature number 262, PWY.5845..superpathway.of.menaquinol.9.biosynthesis
## 2024-11-14 13:24:58.343216 INFO::Fitting model to feature number 263, PWY.6113..superpathway.of.mycolate.biosynthesis
## 2024-11-14 13:24:58.345524 INFO::Fitting model to feature number 264, PWY.5897..superpathway.of.menaquinol.11.biosynthesis
## 2024-11-14 13:24:58.347891 INFO::Fitting model to feature number 265, PWY.5898..superpathway.of.menaquinol.12.biosynthesis
## 2024-11-14 13:24:58.350144 INFO::Fitting model to feature number 266, PWY.5899..superpathway.of.menaquinol.13.biosynthesis
## 2024-11-14 13:24:58.352459 INFO::Fitting model to feature number 267, PWY.7117..C4.photosynthetic.carbon.assimilation.cycle..PEPCK.type
## 2024-11-14 13:24:58.354711 INFO::Fitting model to feature number 268, PWY.6969..TCA.cycle.V..2.oxoglutarate.ferredoxin.oxidoreductase.
## 2024-11-14 13:24:58.357006 INFO::Fitting model to feature number 269, PWY.6628..superpathway.of.L.phenylalanine.biosynthesis
## 2024-11-14 13:24:58.359249 INFO::Fitting model to feature number 270, PWY.5861..superpathway.of.demethylmenaquinol.8.biosynthesis
## 2024-11-14 13:24:58.361565 INFO::Fitting model to feature number 271, PWY.5862..superpathway.of.demethylmenaquinol.9.biosynthesis
## 2024-11-14 13:24:58.36385 INFO::Fitting model to feature number 272, FERMENTATION.PWY..mixed.acid.fermentation
## 2024-11-14 13:24:58.36612 INFO::Fitting model to feature number 273, PWY.241..C4.photosynthetic.carbon.assimilation.cycle..NADP.ME.type
## 2024-11-14 13:24:58.368393 INFO::Fitting model to feature number 274, SO4ASSIM.PWY..sulfate.reduction.I..assimilatory.
## 2024-11-14 13:24:58.370676 INFO::Fitting model to feature number 275, KETOGLUCONMET.PWY..ketogluconate.metabolism
## 2024-11-14 13:24:58.372969 INFO::Fitting model to feature number 276, PWY.5840..superpathway.of.menaquinol.7.biosynthesis
## 2024-11-14 13:24:58.375211 INFO::Fitting model to feature number 277, PWY.5850..superpathway.of.menaquinol.6.biosynthesis.I
## 2024-11-14 13:24:58.377478 INFO::Fitting model to feature number 278, PWY.5896..superpathway.of.menaquinol.10.biosynthesis
## 2024-11-14 13:24:58.379752 INFO::Fitting model to feature number 279, P105.PWY..TCA.cycle.IV..2.oxoglutarate.decarboxylase.
## 2024-11-14 13:24:58.381996 INFO::Fitting model to feature number 280, PWY.5913..TCA.cycle.VI..obligate.autotrophs.
## 2024-11-14 13:24:58.384285 INFO::Fitting model to feature number 281, PWY.561..superpathway.of.glyoxylate.cycle.and.fatty.acid.degradation
## 2024-11-14 13:24:58.386518 INFO::Fitting model to feature number 282, PWY.4702..phytate.degradation.I
## 2024-11-14 13:24:58.388803 INFO::Fitting model to feature number 283, PWY.6803..phosphatidylcholine.acyl.editing
## 2024-11-14 13:24:58.391037 INFO::Fitting model to feature number 284, PWY.7115..C4.photosynthetic.carbon.assimilation.cycle..NAD.ME.type
## 2024-11-14 13:24:58.39334 INFO::Fitting model to feature number 285, PWY.5918..superpathay.of.heme.biosynthesis.from.glutamate
## 2024-11-14 13:24:58.39565 INFO::Fitting model to feature number 286, PWY.7234..inosine.5..phosphate.biosynthesis.III
## 2024-11-14 13:24:58.397884 INFO::Fitting model to feature number 287, PWY.6285..superpathway.of.fatty.acids.biosynthesis..E..coli.
## 2024-11-14 13:24:58.400137 INFO::Fitting model to feature number 288, PWY.5860..superpathway.of.demethylmenaquinol.6.biosynthesis.I
## 2024-11-14 13:24:58.402393 INFO::Fitting model to feature number 289, PWY.5863..superpathway.of.phylloquinol.biosynthesis
## 2024-11-14 13:24:58.404711 INFO::Fitting model to feature number 290, HEME.BIOSYNTHESIS.II..heme.biosynthesis.I..aerobic.
## 2024-11-14 13:24:58.406944 INFO::Fitting model to feature number 291, PWY.821..superpathway.of.sulfur.amino.acid.biosynthesis..Saccharomyces.cerevisiae.
## 2024-11-14 13:24:58.409186 INFO::Fitting model to feature number 292, PWY.5675..nitrate.reduction.V..assimilatory.
## 2024-11-14 13:24:58.411466 INFO::Fitting model to feature number 293, PWY.5791..1.4.dihydroxy.2.naphthoate.biosynthesis.II..plants.
## 2024-11-14 13:24:58.413702 INFO::Fitting model to feature number 294, PWY.5837..1.4.dihydroxy.2.naphthoate.biosynthesis.I
## 2024-11-14 13:24:58.415945 INFO::Fitting model to feature number 295, PWY.5920..superpathway.of.heme.biosynthesis.from.glycine
## 2024-11-14 13:24:58.418171 INFO::Fitting model to feature number 296, PWY.7204..pyridoxal.5..phosphate.salvage.II..plants.
## 2024-11-14 13:24:58.420412 INFO::Fitting model to feature number 297, ENTBACSYN.PWY..enterobactin.biosynthesis
## 2024-11-14 13:24:58.422702 INFO::Fitting model to feature number 298, PWY0.1415..superpathway.of.heme.biosynthesis.from.uroporphyrinogen.III
## 2024-11-14 13:24:58.424946 INFO::Fitting model to feature number 299, HEMESYN2.PWY..heme.biosynthesis.II..anaerobic.
## 2024-11-14 13:24:58.427182 INFO::Fitting model to feature number 300, PWY.6284..superpathway.of.unsaturated.fatty.acids.biosynthesis..E..coli.
## 2024-11-14 13:24:58.429432 INFO::Fitting model to feature number 301, P42.PWY..incomplete.reductive.TCA.cycle
## 2024-11-14 13:24:58.431682 INFO::Fitting model to feature number 302, PWY.5656..mannosylglycerate.biosynthesis.I
## 2024-11-14 13:24:58.433925 INFO::Fitting model to feature number 303, PWY.5173..superpathway.of.acetyl.CoA.biosynthesis
## 2024-11-14 13:24:58.436177 INFO::Fitting model to feature number 304, ECASYN.PWY..enterobacterial.common.antigen.biosynthesis
## 2024-11-14 13:24:58.43839 INFO::Fitting model to feature number 305, AST.PWY..L.arginine.degradation.II..AST.pathway.
## 2024-11-14 13:24:58.44066 INFO::Fitting model to feature number 306, PWY3O.355..stearate.biosynthesis.III..fungi.
## 2024-11-14 13:24:58.442912 INFO::Fitting model to feature number 307, PWY0.42..2.methylcitrate.cycle.I
## 2024-11-14 13:24:58.445167 INFO::Fitting model to feature number 308, PWY.7094..fatty.acid.salvage
## 2024-11-14 13:24:58.447428 INFO::Fitting model to feature number 309, PWY.5747..2.methylcitrate.cycle.II
## 2024-11-14 13:24:58.450148 INFO::Fitting model to feature number 310, PWY.5367..petroselinate.biosynthesis
## 2024-11-14 13:24:58.452405 INFO::Fitting model to feature number 311, PWY0.1533..methylphosphonate.degradation.I
## 2024-11-14 13:24:58.454665 INFO::Fitting model to feature number 312, PWY.6823..molybdenum.cofactor.biosynthesis
## 2024-11-14 13:24:58.456923 INFO::Fitting model to feature number 313, PWY.5705..allantoin.degradation.to.glyoxylate.III
## 2024-11-14 13:24:58.45919 INFO::Fitting model to feature number 314, LPSSYN.PWY..superpathway.of.lipopolysaccharide.biosynthesis
## 2024-11-14 13:24:58.461455 INFO::Fitting model to feature number 315, REDCITCYC..TCA.cycle.VIII..helicobacter.
## 2024-11-14 13:24:58.463725 INFO::Fitting model to feature number 316, GLYCOL.GLYOXDEG.PWY..superpathway.of.glycol.metabolism.and.degradation
## 2024-11-14 13:24:58.465997 INFO::Fitting model to feature number 317, P122.PWY..heterolactic.fermentation
## 2024-11-14 13:24:58.468252 INFO::Fitting model to feature number 318, KDO.NAGLIPASYN.PWY..superpathway.of..Kdo.2.lipid.A.biosynthesis
## 2024-11-14 13:24:58.470546 INFO::Fitting model to feature number 319, PWY.7446..sulfoglycolysis
## 2024-11-14 13:24:58.472891 INFO::Fitting model to feature number 320, PWY490.3..nitrate.reduction.VI..assimilatory.
## 2024-11-14 13:24:58.475167 INFO::Fitting model to feature number 321, PWY.7209..superpathway.of.pyrimidine.ribonucleosides.degradation
## 2024-11-14 13:24:58.477432 INFO::Fitting model to feature number 322, UBISYN.PWY..superpathway.of.ubiquinol.8.biosynthesis..prokaryotic.
## 2024-11-14 13:24:58.479713 INFO::Fitting model to feature number 323, PWY0.41..allantoin.degradation.IV..anaerobic.
## 2024-11-14 13:24:58.481958 INFO::Fitting model to feature number 324, PWY.5855..ubiquinol.7.biosynthesis..prokaryotic.
## 2024-11-14 13:24:58.484244 INFO::Fitting model to feature number 325, PWY.5856..ubiquinol.9.biosynthesis..prokaryotic.
## 2024-11-14 13:24:58.486486 INFO::Fitting model to feature number 326, PWY.5857..ubiquinol.10.biosynthesis..prokaryotic.
## 2024-11-14 13:24:58.488781 INFO::Fitting model to feature number 327, PWY.6708..ubiquinol.8.biosynthesis..prokaryotic.
## 2024-11-14 13:24:58.491031 INFO::Fitting model to feature number 328, PWY.5692..allantoin.degradation.to.glyoxylate.II
## 2024-11-14 13:24:58.493388 INFO::Fitting model to feature number 329, URDEGR.PWY..superpathway.of.allantoin.degradation.in.plants
## 2024-11-14 13:24:58.495682 INFO::Fitting model to feature number 330, PWY0.1338..polymyxin.resistance
## 2024-11-14 13:24:58.497917 INFO::Fitting model to feature number 331, P221.PWY..octane.oxidation
## 2024-11-14 13:24:58.500167 INFO::Fitting model to feature number 332, THREOCAT.PWY..superpathway.of.L.threonine.metabolism
## 2024-11-14 13:24:58.50239 INFO::Fitting model to feature number 333, NONMEVIPP.PWY..methylerythritol.phosphate.pathway.I
## 2024-11-14 13:24:58.504707 INFO::Fitting model to feature number 334, PWY.7560..methylerythritol.phosphate.pathway.II
## 2024-11-14 13:24:58.506943 INFO::Fitting model to feature number 335, PWY.5121..superpathway.of.geranylgeranyl.diphosphate.biosynthesis.II..via.MEP.
## 2024-11-14 13:24:58.509191 INFO::Fitting model to feature number 336, PWY.7392..taxadiene.biosynthesis..engineered.
## 2024-11-14 13:24:58.511451 INFO::Fitting model to feature number 337, PWY.6143..CMP.pseudaminate.biosynthesis
## 2024-11-14 13:24:58.513703 INFO::Fitting model to feature number 338, PWY.6270..isoprene.biosynthesis.I
## 2024-11-14 13:24:58.515979 INFO::Fitting model to feature number 339, ALLANTOINDEG.PWY..superpathway.of.allantoin.degradation.in.yeast
## 2024-11-14 13:24:58.518217 INFO::Fitting model to feature number 340, PWY.6263..superpathway.of.menaquinol.8.biosynthesis.II
## 2024-11-14 13:24:58.520509 INFO::Fitting model to feature number 341, PWY.7371..1.4.dihydroxy.6.naphthoate.biosynthesis.II
## 2024-11-14 13:24:58.522742 INFO::Fitting model to feature number 342, ARGORNPROST.PWY..arginine..ornithine.and.proline.interconversion
## 2024-11-14 13:24:58.525063 INFO::Fitting model to feature number 343, PWY66.398..TCA.cycle.III..animals.
## 2024-11-14 13:24:58.527301 INFO::Fitting model to feature number 344, PWY.5464..superpathway.of.cytosolic.glycolysis..plants...pyruvate.dehydrogenase.and.TCA.cycle
## 2024-11-14 13:24:58.52956 INFO::Fitting model to feature number 345, ORNDEG.PWY..superpathway.of.ornithine.degradation
## 2024-11-14 13:24:58.531804 INFO::Fitting model to feature number 346, P23.PWY..reductive.TCA.cycle.I
## 2024-11-14 13:24:58.534029 INFO::Fitting model to feature number 347, PWY0.1277..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation
## 2024-11-14 13:24:58.5363 INFO::Fitting model to feature number 348, ARGDEG.PWY..superpathway.of.L.arginine..putrescine..and.4.aminobutanoate.degradation
## 2024-11-14 13:24:58.538519 INFO::Fitting model to feature number 349, ORNARGDEG.PWY..superpathway.of.L.arginine.and.L.ornithine.degradation
## 2024-11-14 13:24:58.540763 INFO::Fitting model to feature number 350, HCAMHPDEG.PWY..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation.to.2.oxopent.4.enoate
## 2024-11-14 13:24:58.542981 INFO::Fitting model to feature number 351, PWY.6690..cinnamate.and.3.hydroxycinnamate.degradation.to.2.oxopent.4.enoate
## 2024-11-14 13:24:58.545251 INFO::Fitting model to feature number 352, PROTOCATECHUATE.ORTHO.CLEAVAGE.PWY..protocatechuate.degradation.II..ortho.cleavage.pathway.
## 2024-11-14 13:24:58.547555 INFO::Fitting model to feature number 353, P124.PWY..Bifidobacterium.shunt
## 2024-11-14 13:24:58.549824 INFO::Fitting model to feature number 354, PWY.622..starch.biosynthesis
## 2024-11-14 13:24:58.552112 INFO::Fitting model to feature number 355, PWY.7269..NAD.NADP.NADH.NADPH.mitochondrial.interconversion..yeast.
## 2024-11-14 13:24:58.554371 INFO::Fitting model to feature number 356, PWY.7316..dTDP.N.acetylviosamine.biosynthesis
## 2024-11-14 13:24:58.556615 INFO::Fitting model to feature number 357, PWY.7279..aerobic.respiration.II..cytochrome.c...yeast.
## 2024-11-14 13:24:58.558835 INFO::Fitting model to feature number 358, PWY.6837..fatty.acid.beta.oxidation.V..unsaturated..odd.number..di.isomerase.dependent.
## 2024-11-14 13:24:58.56109 INFO::Fitting model to feature number 359, X3.HYDROXYPHENYLACETATE.DEGRADATION.PWY..4.hydroxyphenylacetate.degradation
## 2024-11-14 13:24:58.563315 INFO::Fitting model to feature number 360, PWY.6953..dTDP.3.acetamido.3.6.dideoxy..alpha..D.galactose.biosynthesis
## 2024-11-14 13:24:58.565585 INFO::Fitting model to feature number 361, PWY.7616..methanol.oxidation.to.carbon.dioxide
## 2024-11-14 13:24:58.567835 INFO::Fitting model to feature number 362, PWY.5088..L.glutamate.degradation.VIII..to.propanoate.
## 2024-11-14 13:24:58.570095 INFO::Fitting model to feature number 363, PWY0.881..superpathway.of.fatty.acid.biosynthesis.I..E..coli.
## 2024-11-14 13:24:58.617015 INFO::Counting total values for each feature
## 2024-11-14 13:24:58.647106 INFO::Writing filtered data to file output_pwys/features/filtered_data.tsv
## 2024-11-14 13:24:58.685525 INFO::Writing filtered, normalized data to file output_pwys/features/filtered_data_norm.tsv
## 2024-11-14 13:24:58.72351 INFO::Writing filtered, normalized, transformed data to file output_pwys/features/filtered_data_norm_transformed.tsv
## 2024-11-14 13:24:58.773022 INFO::Writing residuals to file output_pwys/fits/residuals.rds
## 2024-11-14 13:24:58.796491 INFO::Writing fitted values to file output_pwys/fits/fitted.rds
## 2024-11-14 13:24:58.81263 INFO::Writing all results to file (ordered by increasing q-values): output_pwys/all_results.tsv
## 2024-11-14 13:24:58.818745 INFO::Writing the significant results (those which are less than or equal to the threshold of 0.250000 ) to file (ordered by increasing q-values): output_pwys/significant_results.tsv
## 2024-11-14 13:24:58.819518 INFO::Writing heatmap of significant results to file: output_pwys/heatmap.pdf
## [1] "There are no associations to plot!"
## 2024-11-14 13:24:58.820429 INFO::Writing association plots (one for each significant association) to output folder: output_pwys
## 2024-11-14 13:24:58.823607 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-11-14 13:24:58.824161 INFO::Plotting data for metadata number 1, antibiotics_current_use
## 2024-11-14 13:24:58.825203 INFO::Creating boxplot for categorical data, antibiotics_current_use vs POLYAMINSYN3.PWY..superpathway.of.polyamine.biosynthesis.II
##########################
# Extract the residuals #
##########################
datExpr <- as.data.frame(t(fit_data$residuals))
########################
# Create WGCNA modules #
########################
gsg = goodSamplesGenes(datExpr, verbose = 3)
## Flagging genes and samples with too many missing values...
## ..step 1
## ..Excluding 6 samples from the calculation due to too many missing genes.
## ..step 2
gsg$allOK
## [1] FALSE
if (!gsg$allOK)
{if (sum(!gsg$goodGenes) > 0)
printFlush(paste(
"Removing genes:",
paste(names(datExpr)[!gsg$goodGenes], collapse = ", ")));
if (sum(!gsg$goodSamples) > 0)
printFlush(paste(
"Removing samples:",
paste(rownames(datExpr)[!gsg$goodSamples], collapse =", ")))
datExpr = datExpr[gsg$goodSamples, gsg$goodGenes]
}
## Removing samples: CSM5MCVB_P, CSM79HNY_P, ESM5GEYY_P, ESM718U9_P, MSM6J2N6_P, MSM9VZLX_P
gsg = goodSamplesGenes(datExpr, verbose = 3)
## Flagging genes and samples with too many missing values...
## ..step 1
gsg$allOK # TRUE
## [1] TRUE
###################################
# Choose soft threshold parameter #
###################################
powers = c(c(1:20), seq(from = 22, to=30, by=2))
sft = pickSoftThreshold(
datExpr, powerVector = powers, verbose = 5, dataIsExpr = TRUE,
RsquaredCut = 0.30)
## pickSoftThreshold: will use block size 363.
## pickSoftThreshold: calculating connectivity for given powers...
## ..working on genes 1 through 363 of 363
## Power SFT.R.sq slope truncated.R.sq mean.k. median.k. max.k.
## 1 1 0.722 1.8100 0.7110 124.00 130.000 162.00
## 2 2 0.596 0.6920 0.6290 63.10 68.700 95.70
## 3 3 0.393 0.2300 0.2310 38.40 39.700 69.30
## 4 4 0.124 -0.0902 0.0162 25.90 24.800 55.00
## 5 5 0.561 -0.3550 0.5860 18.70 16.300 46.60
## 6 6 0.673 -0.5190 0.6680 14.20 11.400 40.40
## 7 7 0.781 -0.6230 0.8100 11.20 8.380 35.60
## 8 8 0.815 -0.7040 0.8450 9.07 6.520 31.80
## 9 9 0.861 -0.8180 0.9260 7.54 5.100 28.60
## 10 10 0.847 -0.8620 0.9070 6.39 4.100 26.00
## 11 11 0.835 -0.9150 0.9140 5.50 3.430 23.80
## 12 12 0.847 -0.9540 0.9130 4.80 2.870 21.90
## 13 13 0.868 -0.9730 0.9370 4.24 2.380 20.20
## 14 14 0.864 -0.9990 0.9160 3.78 2.060 18.70
## 15 15 0.890 -1.0300 0.9590 3.40 1.720 17.40
## 16 16 0.898 -1.0400 0.9670 3.09 1.530 16.20
## 17 17 0.888 -1.0600 0.9570 2.82 1.370 15.20
## 18 18 0.877 -1.0600 0.9290 2.59 1.280 14.20
## 19 19 0.864 -1.0500 0.9170 2.40 1.180 13.30
## 20 20 0.844 -1.0600 0.8690 2.23 1.100 12.50
## 21 22 0.854 -1.0700 0.8890 1.95 0.997 11.20
## 22 24 0.820 -1.0600 0.8720 1.74 0.899 9.98
## 23 26 0.797 -0.9790 0.8720 1.57 0.793 8.97
## 24 28 0.829 -0.9390 0.8720 1.43 0.718 8.10
## 25 30 0.807 -0.9130 0.8140 1.31 0.673 7.35
##############################
# One-step module detection #
##############################
power = sft$powerEstimate
net = blockwiseModules(
datExpr,
power = power,
corFnc = "bicor",
corOptions = list(maxPOutliers = 0.1),
networkType ="unsigned",
maxBlockSize = ncol(datExpr),
minModuleSize = 3,
TOMType = "unsigned",
reassignThreshold = 0,
mergeCutHeight = 0,
verbose = 3)
## Calculating module eigengenes block-wise from all genes
## Flagging genes and samples with too many missing values...
## ..step 1
## ..Working on block 1 .
## TOM calculation: adjacency..
## ..will not use multithreading.
## Fraction of slow calculations: 0.000000
## ..connectivity..
## ..matrix multiplication (system BLAS)..
## ..normalization..
## ..done.
## ....clustering..
## ....detecting modules..
## ....calculating module eigengenes..
## ....checking kME in modules..
## ..removing 3 genes from module 1 because their KME is too low.
## ..removing 11 genes from module 2 because their KME is too low.
## ..removing 2 genes from module 3 because their KME is too low.
## ..merging modules that are too close..
## mergeCloseModules: Merging modules whose distance is less than 0
## Calculating new MEs...
####################
# How many modules #
####################
ncol(net$MEs)
## [1] 4
table(net$colors)
##
## blue brown grey turquoise
## 139 18 16 190
##########################
# Plot module dendrogram #
##########################
eigenGenes <- net$MEs
MEDiss = 1-cor(eigenGenes)
METree = hclust(as.dist(MEDiss), method = "average")
plot(METree, main = "Clustering of module eigengenes", xlab = "", sub = "")
###########################################
# Re-calculate modules and find hub genes #
###########################################
moduleColors <- net$colors
MEs0 = moduleEigengenes(datExpr, moduleColors)$eigengenes
modules_data = orderMEs(MEs0)
#######################
# Create mapping file #
#######################
feature_by_modules <- as.data.frame(net$colors)
feature_by_modules <- rownames_to_column(feature_by_modules)
colnames(feature_by_modules) <- c('Feature', 'Module')
features_mapping <- feature_by_modules
features_mapping$Module <- paste('ME', features_mapping$Module, sep = '')We perform the MSEA as before using the modules from the WGCNA analysis on the pathways.
###################
# Rank DA results #
###################
results <- fit_data$results |> filter(metadata=='disease')
results$qval <- p.adjust(results$pval, 'BH')
sum(results$qval < 0.05)
## [1] 0
results <- results[order(results$qval, decreasing = FALSE),]
###################
# MSEA Processing #
##################
module_map <- features_mapping
mod.gs <- tapply(module_map$Module, module_map$Feature, as.character)
microbeSet <- inverseList(mod.gs)
microbeSet
## $MEblue
## [1] "ANAEROFRUCAT.PWY..homolactic.fermentation"
## [2] "ANAGLYCOLYSIS.PWY..glycolysis.III..from.glucose."
## [3] "ARGININE.SYN4.PWY..L.ornithine.de.novo..biosynthesis"
## [4] "ARGSYN.PWY..L.arginine.biosynthesis.I..via.L.ornithine."
## [5] "ARGSYNBSUB.PWY..L.arginine.biosynthesis.II..acetyl.cycle."
## [6] "ARO.PWY..chorismate.biosynthesis.I"
## [7] "ASPASN.PWY..superpathway.of.L.aspartate.and.L.asparagine.biosynthesis"
## [8] "BRANCHED.CHAIN.AA.SYN.PWY..superpathway.of.branched.amino.acid.biosynthesis"
## [9] "CALVIN.PWY..Calvin.Benson.Bassham.cycle"
## [10] "CITRULBIO.PWY..L.citrulline.biosynthesis"
## [11] "COA.PWY..coenzyme.A.biosynthesis.I"
## [12] "COA.PWY.1..coenzyme.A.biosynthesis.II..mammalian."
## [13] "COBALSYN.PWY..adenosylcobalamin.salvage.from.cobinamide.I"
## [14] "COLANSYN.PWY..colanic.acid.building.blocks.biosynthesis"
## [15] "COMPLETE.ARO.PWY..superpathway.of.aromatic.amino.acid.biosynthesis"
## [16] "DENOVOPURINE2.PWY..superpathway.of.purine.nucleotides.de.novo.biosynthesis.II"
## [17] "DTDPRHAMSYN.PWY..dTDP.L.rhamnose.biosynthesis.I"
## [18] "GALACT.GLUCUROCAT.PWY..superpathway.of.hexuronide.and.hexuronate.degradation"
## [19] "GALACTUROCAT.PWY..D.galacturonate.degradation.I"
## [20] "GLUCONEO.PWY..gluconeogenesis.I"
## [21] "GLUCUROCAT.PWY..superpathway.of..beta..D.glucuronide.and.D.glucuronate.degradation"
## [22] "GLUTORN.PWY..L.ornithine.biosynthesis"
## [23] "GLYCOGENSYNTH.PWY..glycogen.biosynthesis.I..from.ADP.D.Glucose."
## [24] "GLYCOLYSIS..glycolysis.I..from.glucose.6.phosphate."
## [25] "HISDEG.PWY..L.histidine.degradation.I"
## [26] "HISTSYN.PWY..L.histidine.biosynthesis"
## [27] "HSERMETANA.PWY..L.methionine.biosynthesis.III"
## [28] "ILEUSYN.PWY..L.isoleucine.biosynthesis.I..from.threonine."
## [29] "NAGLIPASYN.PWY..lipid.IVA.biosynthesis"
## [30] "NONOXIPENT.PWY..pentose.phosphate.pathway..non.oxidative.branch."
## [31] "OANTIGEN.PWY..O.antigen.building.blocks.biosynthesis..E..coli."
## [32] "P108.PWY..pyruvate.fermentation.to.propanoate.I"
## [33] "PANTO.PWY..phosphopantothenate.biosynthesis.I"
## [34] "PANTOSYN.PWY..pantothenate.and.coenzyme.A.biosynthesis.I"
## [35] "PENTOSE.P.PWY..pentose.phosphate.pathway"
## [36] "PEPTIDOGLYCANSYN.PWY..peptidoglycan.biosynthesis.I..meso.diaminopimelate.containing."
## [37] "PHOSLIPSYN.PWY..superpathway.of.phospholipid.biosynthesis.I..bacteria."
## [38] "POLYISOPRENSYN.PWY..polyisoprenoid.biosynthesis..E..coli."
## [39] "PWY.1042..glycolysis.IV..plant.cytosol."
## [40] "PWY.1269..CMP.3.deoxy.D.manno.octulosonate.biosynthesis.I"
## [41] "PWY.2941..L.lysine.biosynthesis.II"
## [42] "PWY.2942..L.lysine.biosynthesis.III"
## [43] "PWY.3001..superpathway.of.L.isoleucine.biosynthesis.I"
## [44] "PWY.3841..folate.transformations.II"
## [45] "PWY.4242..pantothenate.and.coenzyme.A.biosynthesis.III"
## [46] "PWY.4981..L.proline.biosynthesis.II..from.arginine."
## [47] "PWY.4984..urea.cycle"
## [48] "PWY.5030..L.histidine.degradation.III"
## [49] "PWY.5097..L.lysine.biosynthesis.VI"
## [50] "PWY.5103..L.isoleucine.biosynthesis.III"
## [51] "PWY.5154..L.arginine.biosynthesis.III..via.N.acetyl.L.citrulline."
## [52] "PWY.5304..superpathway.of.sulfur.oxidation..Acidianus.ambivalens."
## [53] "PWY.5484..glycolysis.II..from.fructose.6.phosphate."
## [54] "PWY.5505..L.glutamate.and.L.glutamine.biosynthesis"
## [55] "PWY.5659..GDP.mannose.biosynthesis"
## [56] "PWY.5667..CDP.diacylglycerol.biosynthesis.I"
## [57] "PWY.5686..UMP.biosynthesis"
## [58] "PWY.5695..urate.biosynthesis.inosine.5..phosphate.degradation"
## [59] "PWY.5941..glycogen.degradation.II..eukaryotic."
## [60] "PWY.5973..cis.vaccenate.biosynthesis"
## [61] "PWY.6121..5.aminoimidazole.ribonucleotide.biosynthesis.I"
## [62] "PWY.6122..5.aminoimidazole.ribonucleotide.biosynthesis.II"
## [63] "PWY.6123..inosine.5..phosphate.biosynthesis.I"
## [64] "PWY.6124..inosine.5..phosphate.biosynthesis.II"
## [65] "PWY.6125..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.II"
## [66] "PWY.6126..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.II"
## [67] "PWY.6147..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.I"
## [68] "PWY.6151..S.adenosyl.L.methionine.cycle.I"
## [69] "PWY.6163..chorismate.biosynthesis.from.3.dehydroquinate"
## [70] "PWY.6168..flavin.biosynthesis.III..fungi."
## [71] "PWY.621..sucrose.degradation.III..sucrose.invertase."
## [72] "PWY.6277..superpathway.of.5.aminoimidazole.ribonucleotide.biosynthesis"
## [73] "PWY.6305..putrescine.biosynthesis.IV"
## [74] "PWY.6317..galactose.degradation.I..Leloir.pathway."
## [75] "PWY.6385..peptidoglycan.biosynthesis.III..mycobacteria."
## [76] "PWY.6386..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.II..lysine.containing."
## [77] "PWY.6387..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.I..meso.diaminopimelate.containing."
## [78] "PWY.6507..4.deoxy.L.threo.hex.4.enopyranuronate.degradation"
## [79] "PWY.6545..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.III"
## [80] "PWY.6572..chondroitin.sulfate.degradation.I..bacterial."
## [81] "PWY.6609..adenine.and.adenosine.salvage.III"
## [82] "PWY.6700..queuosine.biosynthesis"
## [83] "PWY.6703..preQ0.biosynthesis"
## [84] "PWY.6737..starch.degradation.V"
## [85] "PWY.6859..all.trans.farnesol.biosynthesis"
## [86] "PWY.6892..thiazole.biosynthesis.I..E..coli."
## [87] "PWY.6897..thiamin.salvage.II"
## [88] "PWY.6901..superpathway.of.glucose.and.xylose.degradation"
## [89] "PWY.6936..seleno.amino.acid.biosynthesis"
## [90] "PWY.7111..pyruvate.fermentation.to.isobutanol..engineered."
## [91] "PWY.7184..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.I"
## [92] "PWY.7187..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.II"
## [93] "PWY.7197..pyrimidine.deoxyribonucleotide.phosphorylation"
## [94] "PWY.7199..pyrimidine.deoxyribonucleosides.salvage"
## [95] "PWY.7208..superpathway.of.pyrimidine.nucleobases.salvage"
## [96] "PWY.7210..pyrimidine.deoxyribonucleotides.biosynthesis.from.CTP"
## [97] "PWY.7211..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis"
## [98] "PWY.7219..adenosine.ribonucleotides.de.novo.biosynthesis"
## [99] "PWY.7220..adenosine.deoxyribonucleotides.de.novo.biosynthesis.II"
## [100] "PWY.7221..guanosine.ribonucleotides.de.novo.biosynthesis"
## [101] "PWY.7222..guanosine.deoxyribonucleotides.de.novo.biosynthesis.II"
## [102] "PWY.7228..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.I"
## [103] "PWY.7229..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.I"
## [104] "PWY.7234..inosine.5..phosphate.biosynthesis.III"
## [105] "PWY.724..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.II"
## [106] "PWY.7242..D.fructuronate.degradation"
## [107] "PWY.7282..4.amino.2.methyl.5.phosphomethylpyrimidine.biosynthesis..yeast."
## [108] "PWY.7323..superpathway.of.GDP.mannose.derived.O.antigen.building.blocks.biosynthesis"
## [109] "PWY.7332..superpathway.of.UDP.N.acetylglucosamine.derived.O.antigen.building.blocks.biosynthesis"
## [110] "PWY.7357..thiamin.formation.from.pyrithiamine.and.oxythiamine..yeast."
## [111] "PWY.7383..anaerobic.energy.metabolism..invertebrates..cytosol."
## [112] "PWY.7400..L.arginine.biosynthesis.IV..archaebacteria."
## [113] "PWY.7456..mannan.degradation"
## [114] "PWY.7539..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.III..Chlamydia."
## [115] "PWY.7663..gondoate.biosynthesis..anaerobic."
## [116] "PWY.841..superpathway.of.purine.nucleotides.de.novo.biosynthesis.I"
## [117] "PWY0.1296..purine.ribonucleosides.degradation"
## [118] "PWY0.1319..CDP.diacylglycerol.biosynthesis.II"
## [119] "PWY0.1586..peptidoglycan.maturation..meso.diaminopimelate.containing."
## [120] "PWY0.162..superpathway.of.pyrimidine.ribonucleotides.de.novo.biosynthesis"
## [121] "PWY0.166..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis..E..coli."
## [122] "PWY0.845..superpathway.of.pyridoxal.5..phosphate.biosynthesis.and.salvage"
## [123] "PWY4FS.7..phosphatidylglycerol.biosynthesis.I..plastidic."
## [124] "PWY4FS.8..phosphatidylglycerol.biosynthesis.II..non.plastidic."
## [125] "PWY66.399..gluconeogenesis.III"
## [126] "PWY66.422..D.galactose.degradation.V..Leloir.pathway."
## [127] "PYRIDNUCSYN.PWY..NAD.biosynthesis.I..from.aspartate."
## [128] "PYRIDOXSYN.PWY..pyridoxal.5..phosphate.biosynthesis.I"
## [129] "RHAMCAT.PWY..L.rhamnose.degradation.I"
## [130] "RIBOSYN2.PWY..flavin.biosynthesis.I..bacteria.and.plants."
## [131] "SER.GLYSYN.PWY..superpathway.of.L.serine.and.glycine.biosynthesis.I"
## [132] "TEICHOICACID.PWY..teichoic.acid..poly.glycerol..biosynthesis"
## [133] "THISYN.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.I"
## [134] "THISYNARA.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.III..eukaryotes."
## [135] "THRESYN.PWY..superpathway.of.L.threonine.biosynthesis"
## [136] "TRNA.CHARGING.PWY..tRNA.charging"
## [137] "TRPSYN.PWY..L.tryptophan.biosynthesis"
## [138] "VALSYN.PWY..L.valine.biosynthesis"
## [139] "X1CMET2.PWY..N10.formyl.tetrahydrofolate.biosynthesis"
##
## $MEbrown
## [1] "GLCMANNANAUT.PWY..superpathway.of.N.acetylglucosamine..N.acetylmannosamine.and.N.acetylneuraminate.degradation"
## [2] "GLYCOLYSIS.E.D..superpathway.of.glycolysis.and.Entner.Doudoroff"
## [3] "METH.ACETATE.PWY..methanogenesis.from.acetate"
## [4] "PWY.5100..pyruvate.fermentation.to.acetate.and.lactate.II"
## [5] "PWY.5104..L.isoleucine.biosynthesis.IV"
## [6] "PWY.5177..glutaryl.CoA.degradation"
## [7] "PWY.5690..TCA.cycle.II..plants.and.fungi."
## [8] "PWY.6470..peptidoglycan.biosynthesis.V...beta..lactam.resistance."
## [9] "PWY.6527..stachyose.degradation"
## [10] "PWY.6595..superpathway.of.guanosine.nucleotides.degradation..plants."
## [11] "PWY.6608..guanosine.nucleotides.degradation.III"
## [12] "PWY.7196..superpathway.of.pyrimidine.ribonucleosides.salvage"
## [13] "PWY.7198..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.IV"
## [14] "PWY0.1061..superpathway.of.L.alanine.biosynthesis"
## [15] "PWY0.1261..anhydromuropeptides.recycling"
## [16] "PWY66.400..glycolysis.VI..metazoan."
## [17] "TCA..TCA.cycle.I..prokaryotic."
## [18] "UDPNAGSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.I"
##
## $MEgrey
## [1] "NAD.BIOSYNTHESIS.II..NAD.salvage.pathway.II"
## [2] "P162.PWY..L.glutamate.degradation.V..via.hydroxyglutarate."
## [3] "P163.PWY..L.lysine.fermentation.to.acetate.and.butanoate"
## [4] "PWY.3781..aerobic.respiration.I..cytochrome.c."
## [5] "PWY.6143..CMP.pseudaminate.biosynthesis"
## [6] "PWY.622..starch.biosynthesis"
## [7] "PWY.6263..superpathway.of.menaquinol.8.biosynthesis.II"
## [8] "PWY.6478..GDP.D.glycero..alpha..D.manno.heptose.biosynthesis"
## [9] "PWY.6749..CMP.legionaminate.biosynthesis.I"
## [10] "PWY.6953..dTDP.3.acetamido.3.6.dideoxy..alpha..D.galactose.biosynthesis"
## [11] "PWY.7237..myo...chiro..and.scillo.inositol.degradation"
## [12] "PWY.7312..dTDP.D..beta..fucofuranose.biosynthesis"
## [13] "PWY.7316..dTDP.N.acetylviosamine.biosynthesis"
## [14] "PWY.7371..1.4.dihydroxy.6.naphthoate.biosynthesis.II"
## [15] "PWY490.3..nitrate.reduction.VI..assimilatory."
## [16] "RUMP.PWY..formaldehyde.oxidation.I"
##
## $MEturquoise
## [1] "ALLANTOINDEG.PWY..superpathway.of.allantoin.degradation.in.yeast"
## [2] "ARG.POLYAMINE.SYN..superpathway.of.arginine.and.polyamine.biosynthesis"
## [3] "ARGDEG.PWY..superpathway.of.L.arginine..putrescine..and.4.aminobutanoate.degradation"
## [4] "ARGORNPROST.PWY..arginine..ornithine.and.proline.interconversion"
## [5] "AST.PWY..L.arginine.degradation.II..AST.pathway."
## [6] "BIOTIN.BIOSYNTHESIS.PWY..biotin.biosynthesis.I"
## [7] "CENTFERM.PWY..pyruvate.fermentation.to.butanoate"
## [8] "DAPLYSINESYN.PWY..L.lysine.biosynthesis.I"
## [9] "ECASYN.PWY..enterobacterial.common.antigen.biosynthesis"
## [10] "ENTBACSYN.PWY..enterobactin.biosynthesis"
## [11] "FAO.PWY..fatty.acid..beta..oxidation.I"
## [12] "FASYN.ELONG.PWY..fatty.acid.elongation....saturated"
## [13] "FASYN.INITIAL.PWY..superpathway.of.fatty.acid.biosynthesis.initiation..E..coli."
## [14] "FERMENTATION.PWY..mixed.acid.fermentation"
## [15] "FOLSYN.PWY..superpathway.of.tetrahydrofolate.biosynthesis.and.salvage"
## [16] "FUC.RHAMCAT.PWY..superpathway.of.fucose.and.rhamnose.degradation"
## [17] "FUCCAT.PWY..fucose.degradation"
## [18] "GALACTARDEG.PWY..D.galactarate.degradation.I"
## [19] "GLUCARDEG.PWY..D.glucarate.degradation.I"
## [20] "GLUCARGALACTSUPER.PWY..superpathway.of.D.glucarate.and.D.galactarate.degradation"
## [21] "GLUCOSE1PMETAB.PWY..glucose.and.glucose.1.phosphate.degradation"
## [22] "GLUDEG.I.PWY..GABA.shunt"
## [23] "GLYCOCAT.PWY..glycogen.degradation.I..bacterial."
## [24] "GLYCOL.GLYOXDEG.PWY..superpathway.of.glycol.metabolism.and.degradation"
## [25] "GLYCOLYSIS.TCA.GLYOX.BYPASS..superpathway.of.glycolysis..pyruvate.dehydrogenase..TCA..and.glyoxylate.bypass"
## [26] "GLYOXYLATE.BYPASS..glyoxylate.cycle"
## [27] "GOLPDLCAT.PWY..superpathway.of.glycerol.degradation.to.1.3.propanediol"
## [28] "HCAMHPDEG.PWY..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation.to.2.oxopent.4.enoate"
## [29] "HEME.BIOSYNTHESIS.II..heme.biosynthesis.I..aerobic."
## [30] "HEMESYN2.PWY..heme.biosynthesis.II..anaerobic."
## [31] "HEXITOLDEGSUPER.PWY..superpathway.of.hexitol.degradation..bacteria."
## [32] "HOMOSER.METSYN.PWY..L.methionine.biosynthesis.I"
## [33] "KDO.NAGLIPASYN.PWY..superpathway.of..Kdo.2.lipid.A.biosynthesis"
## [34] "KETOGLUCONMET.PWY..ketogluconate.metabolism"
## [35] "LACTOSECAT.PWY..lactose.and.galactose.degradation.I"
## [36] "LPSSYN.PWY..superpathway.of.lipopolysaccharide.biosynthesis"
## [37] "MET.SAM.PWY..superpathway.of.S.adenosyl.L.methionine.biosynthesis"
## [38] "METHGLYUT.PWY..superpathway.of.methylglyoxal.degradation"
## [39] "METSYN.PWY..L.homoserine.and.L.methionine.biosynthesis"
## [40] "NONMEVIPP.PWY..methylerythritol.phosphate.pathway.I"
## [41] "ORNARGDEG.PWY..superpathway.of.L.arginine.and.L.ornithine.degradation"
## [42] "ORNDEG.PWY..superpathway.of.ornithine.degradation"
## [43] "P105.PWY..TCA.cycle.IV..2.oxoglutarate.decarboxylase."
## [44] "P122.PWY..heterolactic.fermentation"
## [45] "P124.PWY..Bifidobacterium.shunt"
## [46] "P161.PWY..acetylene.degradation"
## [47] "P164.PWY..purine.nucleobases.degradation.I..anaerobic."
## [48] "P185.PWY..formaldehyde.assimilation.III..dihydroxyacetone.cycle."
## [49] "P221.PWY..octane.oxidation"
## [50] "P23.PWY..reductive.TCA.cycle.I"
## [51] "P4.PWY..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.I"
## [52] "P42.PWY..incomplete.reductive.TCA.cycle"
## [53] "P441.PWY..superpathway.of.N.acetylneuraminate.degradation"
## [54] "P461.PWY..hexitol.fermentation.to.lactate..formate..ethanol.and.acetate"
## [55] "POLYAMINSYN3.PWY..superpathway.of.polyamine.biosynthesis.II"
## [56] "POLYAMSYN.PWY..superpathway.of.polyamine.biosynthesis.I"
## [57] "PPGPPMET.PWY..ppGpp.biosynthesis"
## [58] "PROTOCATECHUATE.ORTHO.CLEAVAGE.PWY..protocatechuate.degradation.II..ortho.cleavage.pathway."
## [59] "PRPP.PWY..superpathway.of.histidine..purine..and.pyrimidine.biosynthesis"
## [60] "PWY.1861..formaldehyde.assimilation.II..RuMP.Cycle."
## [61] "PWY.241..C4.photosynthetic.carbon.assimilation.cycle..NADP.ME.type"
## [62] "PWY.2723..trehalose.degradation.V"
## [63] "PWY.4041...gamma..glutamyl.cycle"
## [64] "PWY.4702..phytate.degradation.I"
## [65] "PWY.5005..biotin.biosynthesis.II"
## [66] "PWY.5022..4.aminobutanoate.degradation.V"
## [67] "PWY.5083..NAD.NADH.phosphorylation.and.dephosphorylation"
## [68] "PWY.5088..L.glutamate.degradation.VIII..to.propanoate."
## [69] "PWY.5121..superpathway.of.geranylgeranyl.diphosphate.biosynthesis.II..via.MEP."
## [70] "PWY.5136..fatty.acid..beta..oxidation.II..peroxisome."
## [71] "PWY.5138..unsaturated..even.numbered.fatty.acid..beta..oxidation"
## [72] "PWY.5173..superpathway.of.acetyl.CoA.biosynthesis"
## [73] "PWY.5188..tetrapyrrole.biosynthesis.I..from.glutamate."
## [74] "PWY.5189..tetrapyrrole.biosynthesis.II..from.glycine."
## [75] "PWY.5345..superpathway.of.L.methionine.biosynthesis..by.sulfhydrylation."
## [76] "PWY.5347..superpathway.of.L.methionine.biosynthesis..transsulfuration."
## [77] "PWY.5367..petroselinate.biosynthesis"
## [78] "PWY.5384..sucrose.degradation.IV..sucrose.phosphorylase."
## [79] "PWY.5464..superpathway.of.cytosolic.glycolysis..plants...pyruvate.dehydrogenase.and.TCA.cycle"
## [80] "PWY.561..superpathway.of.glyoxylate.cycle.and.fatty.acid.degradation"
## [81] "PWY.5656..mannosylglycerate.biosynthesis.I"
## [82] "PWY.5675..nitrate.reduction.V..assimilatory."
## [83] "PWY.5676..acetyl.CoA.fermentation.to.butanoate.II"
## [84] "PWY.5677..succinate.fermentation.to.butanoate"
## [85] "PWY.5692..allantoin.degradation.to.glyoxylate.II"
## [86] "PWY.5705..allantoin.degradation.to.glyoxylate.III"
## [87] "PWY.5723..Rubisco.shunt"
## [88] "PWY.5747..2.methylcitrate.cycle.II"
## [89] "PWY.5791..1.4.dihydroxy.2.naphthoate.biosynthesis.II..plants."
## [90] "PWY.5837..1.4.dihydroxy.2.naphthoate.biosynthesis.I"
## [91] "PWY.5838..superpathway.of.menaquinol.8.biosynthesis.I"
## [92] "PWY.5840..superpathway.of.menaquinol.7.biosynthesis"
## [93] "PWY.5845..superpathway.of.menaquinol.9.biosynthesis"
## [94] "PWY.5850..superpathway.of.menaquinol.6.biosynthesis.I"
## [95] "PWY.5855..ubiquinol.7.biosynthesis..prokaryotic."
## [96] "PWY.5856..ubiquinol.9.biosynthesis..prokaryotic."
## [97] "PWY.5857..ubiquinol.10.biosynthesis..prokaryotic."
## [98] "PWY.5860..superpathway.of.demethylmenaquinol.6.biosynthesis.I"
## [99] "PWY.5861..superpathway.of.demethylmenaquinol.8.biosynthesis"
## [100] "PWY.5862..superpathway.of.demethylmenaquinol.9.biosynthesis"
## [101] "PWY.5863..superpathway.of.phylloquinol.biosynthesis"
## [102] "PWY.5896..superpathway.of.menaquinol.10.biosynthesis"
## [103] "PWY.5897..superpathway.of.menaquinol.11.biosynthesis"
## [104] "PWY.5898..superpathway.of.menaquinol.12.biosynthesis"
## [105] "PWY.5899..superpathway.of.menaquinol.13.biosynthesis"
## [106] "PWY.5913..TCA.cycle.VI..obligate.autotrophs."
## [107] "PWY.5918..superpathay.of.heme.biosynthesis.from.glutamate"
## [108] "PWY.5920..superpathway.of.heme.biosynthesis.from.glycine"
## [109] "PWY.5971..palmitate.biosynthesis.II..bacteria.and.plants."
## [110] "PWY.5989..stearate.biosynthesis.II..bacteria.and.plants."
## [111] "PWY.6113..superpathway.of.mycolate.biosynthesis"
## [112] "PWY.6270..isoprene.biosynthesis.I"
## [113] "PWY.6282..palmitoleate.biosynthesis.I..from..5Z..dodec.5.enoate."
## [114] "PWY.6284..superpathway.of.unsaturated.fatty.acids.biosynthesis..E..coli."
## [115] "PWY.6285..superpathway.of.fatty.acids.biosynthesis..E..coli."
## [116] "PWY.6318..L.phenylalanine.degradation.IV..mammalian..via.side.chain."
## [117] "PWY.6353..purine.nucleotides.degradation.II..aerobic."
## [118] "PWY.6471..peptidoglycan.biosynthesis.IV..Enterococcus.faecium."
## [119] "PWY.6519..8.amino.7.oxononanoate.biosynthesis.I"
## [120] "PWY.6531..mannitol.cycle"
## [121] "PWY.6549..L.glutamine.biosynthesis.III"
## [122] "PWY.6588..pyruvate.fermentation.to.acetone"
## [123] "PWY.6590..superpathway.of.Clostridium.acetobutylicum.acidogenic.fermentation"
## [124] "PWY.6606..guanosine.nucleotides.degradation.II"
## [125] "PWY.6612..superpathway.of.tetrahydrofolate.biosynthesis"
## [126] "PWY.6628..superpathway.of.L.phenylalanine.biosynthesis"
## [127] "PWY.6629..superpathway.of.L.tryptophan.biosynthesis"
## [128] "PWY.6630..superpathway.of.L.tyrosine.biosynthesis"
## [129] "PWY.6690..cinnamate.and.3.hydroxycinnamate.degradation.to.2.oxopent.4.enoate"
## [130] "PWY.6708..ubiquinol.8.biosynthesis..prokaryotic."
## [131] "PWY.6731..starch.degradation.III"
## [132] "PWY.6803..phosphatidylcholine.acyl.editing"
## [133] "PWY.6823..molybdenum.cofactor.biosynthesis"
## [134] "PWY.6837..fatty.acid.beta.oxidation.V..unsaturated..odd.number..di.isomerase.dependent."
## [135] "PWY.6876..isopropanol.biosynthesis"
## [136] "PWY.6891..thiazole.biosynthesis.II..Bacillus."
## [137] "PWY.6895..superpathway.of.thiamin.diphosphate.biosynthesis.II"
## [138] "PWY.6969..TCA.cycle.V..2.oxoglutarate.ferredoxin.oxidoreductase."
## [139] "PWY.7003..glycerol.degradation.to.butanol"
## [140] "PWY.7013..L.1.2.propanediol.degradation"
## [141] "PWY.7046..4.coumarate.degradation..anaerobic."
## [142] "PWY.7094..fatty.acid.salvage"
## [143] "PWY.7115..C4.photosynthetic.carbon.assimilation.cycle..NAD.ME.type"
## [144] "PWY.7117..C4.photosynthetic.carbon.assimilation.cycle..PEPCK.type"
## [145] "PWY.7204..pyridoxal.5..phosphate.salvage.II..plants."
## [146] "PWY.7209..superpathway.of.pyrimidine.ribonucleosides.degradation"
## [147] "PWY.7254..TCA.cycle.VII..acetate.producers."
## [148] "PWY.7269..NAD.NADP.NADH.NADPH.mitochondrial.interconversion..yeast."
## [149] "PWY.7279..aerobic.respiration.II..cytochrome.c...yeast."
## [150] "PWY.7288..fatty.acid..beta..oxidation..peroxisome..yeast."
## [151] "PWY.7315..dTDP.N.acetylthomosamine.biosynthesis"
## [152] "PWY.7328..superpathway.of.UDP.glucose.derived.O.antigen.building.blocks.biosynthesis"
## [153] "PWY.7385..1.3.propanediol.biosynthesis..engineered."
## [154] "PWY.7388..octanoyl..acyl.carrier.protein..biosynthesis..mitochondria..yeast."
## [155] "PWY.7392..taxadiene.biosynthesis..engineered."
## [156] "PWY.7446..sulfoglycolysis"
## [157] "PWY.7560..methylerythritol.phosphate.pathway.II"
## [158] "PWY.7616..methanol.oxidation.to.carbon.dioxide"
## [159] "PWY.7664..oleate.biosynthesis.IV..anaerobic."
## [160] "PWY.821..superpathway.of.sulfur.amino.acid.biosynthesis..Saccharomyces.cerevisiae."
## [161] "PWY0.1241..ADP.L.glycero..beta..D.manno.heptose.biosynthesis"
## [162] "PWY0.1277..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation"
## [163] "PWY0.1297..superpathway.of.purine.deoxyribonucleosides.degradation"
## [164] "PWY0.1298..superpathway.of.pyrimidine.deoxyribonucleosides.degradation"
## [165] "PWY0.1338..polymyxin.resistance"
## [166] "PWY0.1415..superpathway.of.heme.biosynthesis.from.uroporphyrinogen.III"
## [167] "PWY0.1479..tRNA.processing"
## [168] "PWY0.1533..methylphosphonate.degradation.I"
## [169] "PWY0.41..allantoin.degradation.IV..anaerobic."
## [170] "PWY0.42..2.methylcitrate.cycle.I"
## [171] "PWY0.781..aspartate.superpathway"
## [172] "PWY0.862...5Z..dodec.5.enoate.biosynthesis"
## [173] "PWY0.881..superpathway.of.fatty.acid.biosynthesis.I..E..coli."
## [174] "PWY3O.355..stearate.biosynthesis.III..fungi."
## [175] "PWY4LZ.257..superpathway.of.fermentation..Chlamydomonas.reinhardtii."
## [176] "PWY66.389..phytol.degradation"
## [177] "PWY66.391..fatty.acid..beta..oxidation.VI..peroxisome."
## [178] "PWY66.398..TCA.cycle.III..animals."
## [179] "PWY66.409..superpathway.of.purine.nucleotide.salvage"
## [180] "PWYG.321..mycolate.biosynthesis"
## [181] "PYRIDNUCSAL.PWY..NAD.salvage.pathway.I"
## [182] "REDCITCYC..TCA.cycle.VIII..helicobacter."
## [183] "SALVADEHYPOX.PWY..adenosine.nucleotides.degradation.II"
## [184] "SO4ASSIM.PWY..sulfate.reduction.I..assimilatory."
## [185] "SULFATE.CYS.PWY..superpathway.of.sulfate.assimilation.and.cysteine.biosynthesis"
## [186] "TCA.GLYOX.BYPASS..superpathway.of.glyoxylate.bypass.and.TCA"
## [187] "THREOCAT.PWY..superpathway.of.L.threonine.metabolism"
## [188] "UBISYN.PWY..superpathway.of.ubiquinol.8.biosynthesis..prokaryotic."
## [189] "URDEGR.PWY..superpathway.of.allantoin.degradation.in.plants"
## [190] "X3.HYDROXYPHENYLACETATE.DEGRADATION.PWY..4.hydroxyphenylacetate.degradation"
############
# Run MSEA #
############
MSEA <- run_MSEA(microbeSet, results$feature)
MSEA <- MSEA[
, c('Set', 'Freq', 'ES', setdiff(names(MSEA), c('Set', 'Freq', 'ES')))]
colnames(MSEA) <- c('ID', 'Size', 'pval', 'qval')
MSEA$ID <- paste(MSEA$ID, ' (', MSEA$Size, ')', sep = '')
########
# Plot #
########
p <- MSEA |>
arrange(-pval) |>
mutate(ID = factor(ID, levels = ID)) |>
ggplot(aes(y = -log10(pval), x = ID)) +
geom_bar(stat = "identity", fill = 'cornflowerblue') + theme_bw() +
coord_flip() +
ggtitle('Statistically significant modules associated with disease') +
xlab('') +
ylab('MSEA enrichment score')
p
Based on the MSEA results, we obtain 4 enriched modules of microbial pathways. We can similarly examine the members of the top enriched modules.
A.5 Print the most significant modules for pathway abundance data
# Print the most significant modules
microbeSet[["MEpurple"]]
## NULL
microbeSet[["MEbrown"]]
## [1] "GLCMANNANAUT.PWY..superpathway.of.N.acetylglucosamine..N.acetylmannosamine.and.N.acetylneuraminate.degradation"
## [2] "GLYCOLYSIS.E.D..superpathway.of.glycolysis.and.Entner.Doudoroff"
## [3] "METH.ACETATE.PWY..methanogenesis.from.acetate"
## [4] "PWY.5100..pyruvate.fermentation.to.acetate.and.lactate.II"
## [5] "PWY.5104..L.isoleucine.biosynthesis.IV"
## [6] "PWY.5177..glutaryl.CoA.degradation"
## [7] "PWY.5690..TCA.cycle.II..plants.and.fungi."
## [8] "PWY.6470..peptidoglycan.biosynthesis.V...beta..lactam.resistance."
## [9] "PWY.6527..stachyose.degradation"
## [10] "PWY.6595..superpathway.of.guanosine.nucleotides.degradation..plants."
## [11] "PWY.6608..guanosine.nucleotides.degradation.III"
## [12] "PWY.7196..superpathway.of.pyrimidine.ribonucleosides.salvage"
## [13] "PWY.7198..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.IV"
## [14] "PWY0.1061..superpathway.of.L.alanine.biosynthesis"
## [15] "PWY0.1261..anhydromuropeptides.recycling"
## [16] "PWY66.400..glycolysis.VI..metazoan."
## [17] "TCA..TCA.cycle.I..prokaryotic."
## [18] "UDPNAGSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.I"