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"
## 2025-02-26 12:08:07.385163 INFO::Writing function arguments to log file
## 2025-02-26 12:08:07.400189 INFO::Verifying options selected are valid
## 2025-02-26 12:08:07.427169 INFO::Determining format of input files
## 2025-02-26 12:08:07.42778 INFO::Input format is data samples as rows and metadata samples as rows
## 2025-02-26 12:08:07.433578 INFO::Formula for fixed effects: expr ~ disease + age + antibiotics_current_use
## 2025-02-26 12:08:07.434271 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
## 2025-02-26 12:08:07.434897 INFO::Filter data based on min abundance and min prevalence
## 2025-02-26 12:08:07.435343 INFO::Total samples in data: 136
## 2025-02-26 12:08:07.435782 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
## 2025-02-26 12:08:07.441551 INFO::Total filtered features: 452
## 2025-02-26 12:08:07.442291 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
## 2025-02-26 12:08:07.445066 INFO::Total filtered features with variance filtering: 0
## 2025-02-26 12:08:07.445588 INFO::Filtered feature names from variance filtering:
## 2025-02-26 12:08:07.446049 INFO::Running selected normalization method: NONE
## 2025-02-26 12:08:07.446521 INFO::Applying z-score to standardize continuous metadata
## 2025-02-26 12:08:07.450697 INFO::Running selected transform method: LOG
## 2025-02-26 12:08:07.453563 INFO::Running selected analysis method: LM
## 2025-02-26 12:08:07.457147 INFO::Fitting model to feature number 1, Acidaminococcus.intestini
## 2025-02-26 12:08:07.463191 INFO::Fitting model to feature number 2, Agathobaculum.butyriciproducens
## 2025-02-26 12:08:07.46552 INFO::Fitting model to feature number 3, Akkermansia.muciniphila
## 2025-02-26 12:08:07.467837 INFO::Fitting model to feature number 4, Alistipes.finegoldii
## 2025-02-26 12:08:07.470146 INFO::Fitting model to feature number 5, Alistipes.indistinctus
## 2025-02-26 12:08:07.472435 INFO::Fitting model to feature number 6, Alistipes.putredinis
## 2025-02-26 12:08:07.474705 INFO::Fitting model to feature number 7, Alistipes.shahii
## 2025-02-26 12:08:07.477004 INFO::Fitting model to feature number 8, Anaerobutyricum.hallii
## 2025-02-26 12:08:07.479259 INFO::Fitting model to feature number 9, Anaeromassilibacillus.sp..An250
## 2025-02-26 12:08:07.481606 INFO::Fitting model to feature number 10, Anaerostipes.hadrus
## 2025-02-26 12:08:07.483895 INFO::Fitting model to feature number 11, Anaerotignum.lactatifermentans
## 2025-02-26 12:08:07.486176 INFO::Fitting model to feature number 12, Anaerotruncus.colihominis
## 2025-02-26 12:08:07.488435 INFO::Fitting model to feature number 13, Bacteroides.caccae
## 2025-02-26 12:08:07.490687 INFO::Fitting model to feature number 14, Bacteroides.cellulosilyticus
## 2025-02-26 12:08:07.492956 INFO::Fitting model to feature number 15, Bacteroides.eggerthii
## 2025-02-26 12:08:07.495221 INFO::Fitting model to feature number 16, Bacteroides.faecis
## 2025-02-26 12:08:07.497481 INFO::Fitting model to feature number 17, Bacteroides.faecis.CAG.32
## 2025-02-26 12:08:07.499758 INFO::Fitting model to feature number 18, Bacteroides.finegoldii
## 2025-02-26 12:08:07.502077 INFO::Fitting model to feature number 19, Bacteroides.fragilis
## 2025-02-26 12:08:07.504385 INFO::Fitting model to feature number 20, Bacteroides.galacturonicus
## 2025-02-26 12:08:07.506639 INFO::Fitting model to feature number 21, Bacteroides.intestinalis
## 2025-02-26 12:08:07.508928 INFO::Fitting model to feature number 22, Bacteroides.ovatus
## 2025-02-26 12:08:07.511187 INFO::Fitting model to feature number 23, Bacteroides.salyersiae
## 2025-02-26 12:08:07.513446 INFO::Fitting model to feature number 24, Bacteroides.stercoris
## 2025-02-26 12:08:07.51575 INFO::Fitting model to feature number 25, Bacteroides.thetaiotaomicron
## 2025-02-26 12:08:07.518015 INFO::Fitting model to feature number 26, Bacteroides.uniformis
## 2025-02-26 12:08:07.520295 INFO::Fitting model to feature number 27, Bacteroides.xylanisolvens
## 2025-02-26 12:08:07.522552 INFO::Fitting model to feature number 28, Barnesiella.intestinihominis
## 2025-02-26 12:08:07.524835 INFO::Fitting model to feature number 29, Bifidobacterium.adolescentis
## 2025-02-26 12:08:07.52723 INFO::Fitting model to feature number 30, Bifidobacterium.bifidum
## 2025-02-26 12:08:07.529484 INFO::Fitting model to feature number 31, Bifidobacterium.longum
## 2025-02-26 12:08:07.531773 INFO::Fitting model to feature number 32, Bifidobacterium.pseudocatenulatum
## 2025-02-26 12:08:07.534042 INFO::Fitting model to feature number 33, Bilophila.wadsworthia
## 2025-02-26 12:08:07.536313 INFO::Fitting model to feature number 34, Blautia.obeum
## 2025-02-26 12:08:07.538573 INFO::Fitting model to feature number 35, Blautia.sp..CAG.257
## 2025-02-26 12:08:07.540872 INFO::Fitting model to feature number 36, Blautia.wexlerae
## 2025-02-26 12:08:07.543142 INFO::Fitting model to feature number 37, Butyricimonas.synergistica
## 2025-02-26 12:08:07.545424 INFO::Fitting model to feature number 38, Butyricimonas.virosa
## 2025-02-26 12:08:07.547746 INFO::Fitting model to feature number 39, Clostridium.bolteae.CAG.59
## 2025-02-26 12:08:07.550038 INFO::Fitting model to feature number 40, Clostridium.sp..CAG.299
## 2025-02-26 12:08:07.5523 INFO::Fitting model to feature number 41, Clostridium.sp..CAG.58
## 2025-02-26 12:08:07.554558 INFO::Fitting model to feature number 42, Collinsella.aerofaciens
## 2025-02-26 12:08:07.556826 INFO::Fitting model to feature number 43, Collinsella.intestinalis
## 2025-02-26 12:08:07.559097 INFO::Fitting model to feature number 44, Coprobacter.fastidiosus
## 2025-02-26 12:08:07.561427 INFO::Fitting model to feature number 45, Coprococcus.catus
## 2025-02-26 12:08:07.563813 INFO::Fitting model to feature number 46, Coprococcus.comes
## 2025-02-26 12:08:07.566089 INFO::Fitting model to feature number 47, Coprococcus.eutactus
## 2025-02-26 12:08:07.568369 INFO::Fitting model to feature number 48, Dialister.invisus
## 2025-02-26 12:08:07.570649 INFO::Fitting model to feature number 49, Dielma.fastidiosa
## 2025-02-26 12:08:07.572936 INFO::Fitting model to feature number 50, Dorea.formicigenerans
## 2025-02-26 12:08:07.575217 INFO::Fitting model to feature number 51, Dorea.longicatena
## 2025-02-26 12:08:07.577483 INFO::Fitting model to feature number 52, Eggerthella.lenta
## 2025-02-26 12:08:07.579765 INFO::Fitting model to feature number 53, Eisenbergiella.massiliensis
## 2025-02-26 12:08:07.58204 INFO::Fitting model to feature number 54, Eisenbergiella.tayi
## 2025-02-26 12:08:07.584315 INFO::Fitting model to feature number 55, Enterocloster.aldenensis
## 2025-02-26 12:08:07.586601 INFO::Fitting model to feature number 56, Enterocloster.bolteae
## 2025-02-26 12:08:07.588889 INFO::Fitting model to feature number 57, Enterocloster.citroniae
## 2025-02-26 12:08:07.59117 INFO::Fitting model to feature number 58, Enterocloster.clostridioformis
## 2025-02-26 12:08:07.593447 INFO::Fitting model to feature number 59, Enterocloster.lavalensis
## 2025-02-26 12:08:07.595762 INFO::Fitting model to feature number 60, Erysipelatoclostridium.ramosum
## 2025-02-26 12:08:07.59802 INFO::Fitting model to feature number 61, Escherichia.coli
## 2025-02-26 12:08:07.600366 INFO::Fitting model to feature number 62, Eubacterium.ramulus
## 2025-02-26 12:08:07.602648 INFO::Fitting model to feature number 63, Eubacterium.sp..CAG.251
## 2025-02-26 12:08:07.604941 INFO::Fitting model to feature number 64, Eubacterium.sp..CAG.38
## 2025-02-26 12:08:07.607208 INFO::Fitting model to feature number 65, Eubacterium.ventriosum
## 2025-02-26 12:08:07.609475 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2025-02-26 12:08:07.61184 INFO::Fitting model to feature number 67, Firmicutes.bacterium.CAG.83
## 2025-02-26 12:08:07.614125 INFO::Fitting model to feature number 68, Firmicutes.bacterium.CAG.94
## 2025-02-26 12:08:07.616388 INFO::Fitting model to feature number 69, Flavonifractor.plautii
## 2025-02-26 12:08:07.61865 INFO::Fitting model to feature number 70, Fusicatenibacter.saccharivorans
## 2025-02-26 12:08:07.62093 INFO::Fitting model to feature number 71, Gemmiger.formicilis
## 2025-02-26 12:08:07.623194 INFO::Fitting model to feature number 72, Haemophilus.parainfluenzae
## 2025-02-26 12:08:07.625476 INFO::Fitting model to feature number 73, Haemophilus.sp..HMSC71H05
## 2025-02-26 12:08:07.627743 INFO::Fitting model to feature number 74, Holdemania.filiformis
## 2025-02-26 12:08:07.630024 INFO::Fitting model to feature number 75, Hungatella.hathewayi
## 2025-02-26 12:08:07.632279 INFO::Fitting model to feature number 76, Intestinibacter.bartlettii
## 2025-02-26 12:08:07.634562 INFO::Fitting model to feature number 77, Intestinimonas.butyriciproducens
## 2025-02-26 12:08:07.636846 INFO::Fitting model to feature number 78, Klebsiella.pneumoniae
## 2025-02-26 12:08:07.639113 INFO::Fitting model to feature number 79, Klebsiella.variicola
## 2025-02-26 12:08:07.641423 INFO::Fitting model to feature number 80, Lachnospira.eligens
## 2025-02-26 12:08:07.643699 INFO::Fitting model to feature number 81, Lachnospira.pectinoschiza
## 2025-02-26 12:08:07.646001 INFO::Fitting model to feature number 82, Lacrimispora.saccharolytica
## 2025-02-26 12:08:07.64829 INFO::Fitting model to feature number 83, Lactobacillus.rogosae
## 2025-02-26 12:08:07.650555 INFO::Fitting model to feature number 84, Lawsonibacter.asaccharolyticus
## 2025-02-26 12:08:07.652853 INFO::Fitting model to feature number 85, Monoglobus.pectinilyticus
## 2025-02-26 12:08:07.655123 INFO::Fitting model to feature number 86, Odoribacter.splanchnicus
## 2025-02-26 12:08:07.657407 INFO::Fitting model to feature number 87, Oscillibacter.sp..57_20
## 2025-02-26 12:08:07.659685 INFO::Fitting model to feature number 88, Oscillibacter.sp..CAG.241
## 2025-02-26 12:08:07.661968 INFO::Fitting model to feature number 89, Parabacteroides.distasonis
## 2025-02-26 12:08:07.664336 INFO::Fitting model to feature number 90, Parabacteroides.merdae
## 2025-02-26 12:08:07.66661 INFO::Fitting model to feature number 91, Paraprevotella.clara
## 2025-02-26 12:08:07.668919 INFO::Fitting model to feature number 92, Paraprevotella.xylaniphila
## 2025-02-26 12:08:07.671211 INFO::Fitting model to feature number 93, Parasutterella.excrementihominis
## 2025-02-26 12:08:07.673502 INFO::Fitting model to feature number 94, Phascolarctobacterium.faecium
## 2025-02-26 12:08:07.67581 INFO::Fitting model to feature number 95, Phocaeicola.coprocola
## 2025-02-26 12:08:07.678201 INFO::Fitting model to feature number 96, Phocaeicola.dorei
## 2025-02-26 12:08:07.680522 INFO::Fitting model to feature number 97, Phocaeicola.massiliensis
## 2025-02-26 12:08:07.682827 INFO::Fitting model to feature number 98, Phocaeicola.plebeius
## 2025-02-26 12:08:07.685105 INFO::Fitting model to feature number 99, Phocaeicola.vulgatus
## 2025-02-26 12:08:07.687396 INFO::Fitting model to feature number 100, Prevotella.copri
## 2025-02-26 12:08:07.689663 INFO::Fitting model to feature number 101, Proteobacteria.bacterium.CAG.139
## 2025-02-26 12:08:07.691942 INFO::Fitting model to feature number 102, Roseburia.faecis
## 2025-02-26 12:08:07.694209 INFO::Fitting model to feature number 103, Roseburia.hominis
## 2025-02-26 12:08:07.696498 INFO::Fitting model to feature number 104, Roseburia.intestinalis
## 2025-02-26 12:08:07.698768 INFO::Fitting model to feature number 105, Roseburia.inulinivorans
## 2025-02-26 12:08:07.701024 INFO::Fitting model to feature number 106, Roseburia.sp..CAG.471
## 2025-02-26 12:08:07.703281 INFO::Fitting model to feature number 107, Ruminococcaceae.bacterium.D16
## 2025-02-26 12:08:07.705533 INFO::Fitting model to feature number 108, Ruminococcus.bicirculans
## 2025-02-26 12:08:07.707798 INFO::Fitting model to feature number 109, Ruminococcus.bromii
## 2025-02-26 12:08:07.71006 INFO::Fitting model to feature number 110, Ruthenibacterium.lactatiformans
## 2025-02-26 12:08:07.712331 INFO::Fitting model to feature number 111, Sellimonas.intestinalis
## 2025-02-26 12:08:07.714614 INFO::Fitting model to feature number 112, Streptococcus.parasanguinis
## 2025-02-26 12:08:07.716906 INFO::Fitting model to feature number 113, Streptococcus.salivarius
## 2025-02-26 12:08:07.719209 INFO::Fitting model to feature number 114, Turicimonas.muris
## 2025-02-26 12:08:07.721543 INFO::Fitting model to feature number 115, Veillonella.atypica
## 2025-02-26 12:08:07.723879 INFO::Fitting model to feature number 116, Veillonella.dispar
## 2025-02-26 12:08:07.726181 INFO::Fitting model to feature number 117, Veillonella.infantium
## 2025-02-26 12:08:07.728465 INFO::Fitting model to feature number 118, Veillonella.parvula
## 2025-02-26 12:08:07.730776 INFO::Fitting model to feature number 119, Veillonella.sp..T11011.6
## 2025-02-26 12:08:07.733135 INFO::Fitting model to feature number 120, X.Clostridium..innocuum
## 2025-02-26 12:08:07.735414 INFO::Fitting model to feature number 121, X.Clostridium..leptum
## 2025-02-26 12:08:07.737679 INFO::Fitting model to feature number 122, X.Clostridium..symbiosum
## 2025-02-26 12:08:07.740007 INFO::Fitting model to feature number 123, X.Eubacterium..rectale
## 2025-02-26 12:08:07.742293 INFO::Fitting model to feature number 124, X.Eubacterium..siraeum
## 2025-02-26 12:08:07.744579 INFO::Fitting model to feature number 125, X.Ruminococcus..gnavus
## 2025-02-26 12:08:07.746983 INFO::Fitting model to feature number 126, X.Ruminococcus..lactaris
## 2025-02-26 12:08:07.749402 INFO::Fitting model to feature number 127, X.Ruminococcus..torques
## 2025-02-26 12:08:07.770009 INFO::Counting total values for each feature
## 2025-02-26 12:08:07.7814 INFO::Writing filtered data to file output_species/features/filtered_data.tsv
## 2025-02-26 12:08:07.790321 INFO::Writing filtered, normalized data to file output_species/features/filtered_data_norm.tsv
## 2025-02-26 12:08:07.798803 INFO::Writing filtered, normalized, transformed data to file output_species/features/filtered_data_norm_transformed.tsv
## 2025-02-26 12:08:07.811688 INFO::Writing residuals to file output_species/fits/residuals.rds
## 2025-02-26 12:08:07.817289 INFO::Writing fitted values to file output_species/fits/fitted.rds
## 2025-02-26 12:08:07.822095 INFO::Writing all results to file (ordered by increasing q-values): output_species/all_results.tsv
## 2025-02-26 12:08:07.824554 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
## 2025-02-26 12:08:07.825448 INFO::Writing heatmap of significant results to file: output_species/heatmap.pdf
## 2025-02-26 12:08:07.935611 INFO::Writing association plots (one for each significant association) to output folder: output_species
## 2025-02-26 12:08:07.938479 INFO::Plotting associations from most to least significant, grouped by metadata
## 2025-02-26 12:08:07.939116 INFO::Plotting data for metadata number 1, age
## 2025-02-26 12:08:07.940094 INFO::Creating scatter plot for continuous data, age vs Alistipes.indistinctus
## 2025-02-26 12:08:08.09795 INFO::Creating scatter plot for continuous data, age vs Lacrimispora.saccharolytica
## 2025-02-26 12:08:08.301522 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bicirculans
## 2025-02-26 12:08:08.42708 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
## 2025-02-26 12:08:08.556216 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
## 2025-02-26 12:08:08.689868 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
## 2025-02-26 12:08:08.84243 INFO::Creating scatter plot for continuous data, age vs Lachnospira.eligens
## 2025-02-26 12:08:08.975263 INFO::Creating scatter plot for continuous data, age vs X.Ruminococcus..gnavus
## 2025-02-26 12:08:09.118557 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.virosa
## 2025-02-26 12:08:09.250614 INFO::Creating scatter plot for continuous data, age vs Lawsonibacter.asaccharolyticus
## 2025-02-26 12:08:09.394654 INFO::Creating scatter plot for continuous data, age vs X.Eubacterium..siraeum
## 2025-02-26 12:08:09.522358 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.synergistica
## 2025-02-26 12:08:09.650965 INFO::Creating scatter plot for continuous data, age vs Monoglobus.pectinilyticus
## 2025-02-26 12:08:09.780551 INFO::Creating scatter plot for continuous data, age vs Veillonella.atypica
## 2025-02-26 12:08:11.414834 INFO::Plotting data for metadata number 2, disease
## 2025-02-26 12:08:11.41625 INFO::Creating boxplot for categorical data, disease vs Alistipes.putredinis
## 2025-02-26 12:08:11.586766 INFO::Creating boxplot for categorical data, disease vs Gemmiger.formicilis
## 2025-02-26 12:08:11.720712 INFO::Creating boxplot for categorical data, disease vs X.Ruminococcus..torques
## 2025-02-26 12:08:11.842757 INFO::Creating boxplot for categorical data, disease vs Ruminococcus.bicirculans
## 2025-02-26 12:08:11.961198 INFO::Creating boxplot for categorical data, disease vs Sellimonas.intestinalis
## 2025-02-26 12:08:12.078452 INFO::Creating boxplot for categorical data, disease vs X.Clostridium..leptum
## 2025-02-26 12:08:12.211135 INFO::Creating boxplot for categorical data, disease vs Alistipes.shahii
## 2025-02-26 12:08:13.312285 INFO::Plotting data for metadata number 3, antibiotics_current_use
## 2025-02-26 12:08:13.313618 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Coprobacter.fastidiosus
## 2025-02-26 12:08:13.431788 INFO::Creating boxplot for categorical data, antibiotics_current_use vs X.Eubacterium..rectale
## 2025-02-26 12:08:13.549145 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"
## 2025-02-26 12:08:38.623485 INFO::Writing function arguments to log file
## 2025-02-26 12:08:38.627313 INFO::Verifying options selected are valid
## 2025-02-26 12:08:38.627809 INFO::Determining format of input files
## 2025-02-26 12:08:38.628303 INFO::Input format is data samples as rows and metadata samples as rows
## 2025-02-26 12:08:38.633818 INFO::Formula for fixed effects: expr ~ disease + age + antibiotics_current_use
## 2025-02-26 12:08:38.634372 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
## 2025-02-26 12:08:38.634989 INFO::Filter data based on min abundance and min prevalence
## 2025-02-26 12:08:38.635441 INFO::Total samples in data: 136
## 2025-02-26 12:08:38.635885 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
## 2025-02-26 12:08:38.641222 INFO::Total filtered features: 113
## 2025-02-26 12:08:38.641834 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
## 2025-02-26 12:08:38.648011 INFO::Total filtered features with variance filtering: 0
## 2025-02-26 12:08:38.648555 INFO::Filtered feature names from variance filtering:
## 2025-02-26 12:08:38.649015 INFO::Running selected normalization method: NONE
## 2025-02-26 12:08:38.649425 INFO::Applying z-score to standardize continuous metadata
## 2025-02-26 12:08:38.652942 INFO::Running selected transform method: LOG
## 2025-02-26 12:08:38.660713 INFO::Running selected analysis method: LM
## 2025-02-26 12:08:38.661286 INFO::Fitting model to feature number 1, PWY.1042..glycolysis.IV..plant.cytosol.
## 2025-02-26 12:08:38.663975 INFO::Fitting model to feature number 2, DTDPRHAMSYN.PWY..dTDP.L.rhamnose.biosynthesis.I
## 2025-02-26 12:08:38.666332 INFO::Fitting model to feature number 3, PWY.5695..urate.biosynthesis.inosine.5..phosphate.degradation
## 2025-02-26 12:08:38.668655 INFO::Fitting model to feature number 4, PWY.6936..seleno.amino.acid.biosynthesis
## 2025-02-26 12:08:38.671021 INFO::Fitting model to feature number 5, ILEUSYN.PWY..L.isoleucine.biosynthesis.I..from.threonine.
## 2025-02-26 12:08:38.673347 INFO::Fitting model to feature number 6, PWY.7111..pyruvate.fermentation.to.isobutanol..engineered.
## 2025-02-26 12:08:38.675672 INFO::Fitting model to feature number 7, VALSYN.PWY..L.valine.biosynthesis
## 2025-02-26 12:08:38.678016 INFO::Fitting model to feature number 8, PWY.6609..adenine.and.adenosine.salvage.III
## 2025-02-26 12:08:38.680329 INFO::Fitting model to feature number 9, PWY.6737..starch.degradation.V
## 2025-02-26 12:08:38.682701 INFO::Fitting model to feature number 10, PWY.7219..adenosine.ribonucleotides.de.novo.biosynthesis
## 2025-02-26 12:08:38.685057 INFO::Fitting model to feature number 11, PWY0.1586..peptidoglycan.maturation..meso.diaminopimelate.containing.
## 2025-02-26 12:08:38.687426 INFO::Fitting model to feature number 12, ASPASN.PWY..superpathway.of.L.aspartate.and.L.asparagine.biosynthesis
## 2025-02-26 12:08:38.689785 INFO::Fitting model to feature number 13, PWY.5973..cis.vaccenate.biosynthesis
## 2025-02-26 12:08:38.69211 INFO::Fitting model to feature number 14, PWY.7221..guanosine.ribonucleotides.de.novo.biosynthesis
## 2025-02-26 12:08:38.694425 INFO::Fitting model to feature number 15, PWY.5686..UMP.biosynthesis
## 2025-02-26 12:08:38.696776 INFO::Fitting model to feature number 16, X1CMET2.PWY..N10.formyl.tetrahydrofolate.biosynthesis
## 2025-02-26 12:08:38.699114 INFO::Fitting model to feature number 17, PWY.6122..5.aminoimidazole.ribonucleotide.biosynthesis.II
## 2025-02-26 12:08:38.701431 INFO::Fitting model to feature number 18, PWY.6277..superpathway.of.5.aminoimidazole.ribonucleotide.biosynthesis
## 2025-02-26 12:08:38.703767 INFO::Fitting model to feature number 19, PWY.6385..peptidoglycan.biosynthesis.III..mycobacteria.
## 2025-02-26 12:08:38.706069 INFO::Fitting model to feature number 20, PWY.3841..folate.transformations.II
## 2025-02-26 12:08:38.708375 INFO::Fitting model to feature number 21, RIBOSYN2.PWY..flavin.biosynthesis.I..bacteria.and.plants.
## 2025-02-26 12:08:38.71069 INFO::Fitting model to feature number 22, PWY.6387..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.I..meso.diaminopimelate.containing.
## 2025-02-26 12:08:38.713042 INFO::Fitting model to feature number 23, THISYNARA.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.III..eukaryotes.
## 2025-02-26 12:08:38.715352 INFO::Fitting model to feature number 24, PWY.7663..gondoate.biosynthesis..anaerobic.
## 2025-02-26 12:08:38.717677 INFO::Fitting model to feature number 25, PWY.6386..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.II..lysine.containing.
## 2025-02-26 12:08:38.720061 INFO::Fitting model to feature number 26, PWY.6700..queuosine.biosynthesis
## 2025-02-26 12:08:38.722385 INFO::Fitting model to feature number 27, PEPTIDOGLYCANSYN.PWY..peptidoglycan.biosynthesis.I..meso.diaminopimelate.containing.
## 2025-02-26 12:08:38.724688 INFO::Fitting model to feature number 28, TRNA.CHARGING.PWY..tRNA.charging
## 2025-02-26 12:08:38.727071 INFO::Fitting model to feature number 29, PWY.6121..5.aminoimidazole.ribonucleotide.biosynthesis.I
## 2025-02-26 12:08:38.729385 INFO::Fitting model to feature number 30, HISTSYN.PWY..L.histidine.biosynthesis
## 2025-02-26 12:08:38.731707 INFO::Fitting model to feature number 31, PWY.7229..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.I
## 2025-02-26 12:08:38.734078 INFO::Fitting model to feature number 32, PWY.7199..pyrimidine.deoxyribonucleosides.salvage
## 2025-02-26 12:08:38.7364 INFO::Fitting model to feature number 33, PANTO.PWY..phosphopantothenate.biosynthesis.I
## 2025-02-26 12:08:38.738773 INFO::Fitting model to feature number 34, PWY.2942..L.lysine.biosynthesis.III
## 2025-02-26 12:08:38.741105 INFO::Fitting model to feature number 35, PWY.7237..myo...chiro..and.scillo.inositol.degradation
## 2025-02-26 12:08:38.743429 INFO::Fitting model to feature number 36, PWY.6168..flavin.biosynthesis.III..fungi.
## 2025-02-26 12:08:38.745876 INFO::Fitting model to feature number 37, COA.PWY.1..coenzyme.A.biosynthesis.II..mammalian.
## 2025-02-26 12:08:38.748226 INFO::Fitting model to feature number 38, PWY.5667..CDP.diacylglycerol.biosynthesis.I
## 2025-02-26 12:08:38.750573 INFO::Fitting model to feature number 39, PWY0.1319..CDP.diacylglycerol.biosynthesis.II
## 2025-02-26 12:08:38.75292 INFO::Fitting model to feature number 40, PWY.5097..L.lysine.biosynthesis.VI
## 2025-02-26 12:08:38.755245 INFO::Fitting model to feature number 41, ANAGLYCOLYSIS.PWY..glycolysis.III..from.glucose.
## 2025-02-26 12:08:38.757559 INFO::Fitting model to feature number 42, PWY.6123..inosine.5..phosphate.biosynthesis.I
## 2025-02-26 12:08:38.759911 INFO::Fitting model to feature number 43, ARGININE.SYN4.PWY..L.ornithine.de.novo..biosynthesis
## 2025-02-26 12:08:38.762231 INFO::Fitting model to feature number 44, PWY.6163..chorismate.biosynthesis.from.3.dehydroquinate
## 2025-02-26 12:08:38.764554 INFO::Fitting model to feature number 45, THRESYN.PWY..superpathway.of.L.threonine.biosynthesis
## 2025-02-26 12:08:38.766916 INFO::Fitting model to feature number 46, PYRIDNUCSYN.PWY..NAD.biosynthesis.I..from.aspartate.
## 2025-02-26 12:08:38.769235 INFO::Fitting model to feature number 47, PWY.6124..inosine.5..phosphate.biosynthesis.II
## 2025-02-26 12:08:38.771679 INFO::Fitting model to feature number 48, PWY.6147..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.I
## 2025-02-26 12:08:38.774054 INFO::Fitting model to feature number 49, PWY.7539..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.III..Chlamydia.
## 2025-02-26 12:08:38.776616 INFO::Fitting model to feature number 50, PWY.6151..S.adenosyl.L.methionine.cycle.I
## 2025-02-26 12:08:38.779015 INFO::Fitting model to feature number 51, SER.GLYSYN.PWY..superpathway.of.L.serine.and.glycine.biosynthesis.I
## 2025-02-26 12:08:38.781332 INFO::Fitting model to feature number 52, PWY.6126..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.II
## 2025-02-26 12:08:38.783662 INFO::Fitting model to feature number 53, PANTOSYN.PWY..pantothenate.and.coenzyme.A.biosynthesis.I
## 2025-02-26 12:08:38.786027 INFO::Fitting model to feature number 54, PWY.7228..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.I
## 2025-02-26 12:08:38.78835 INFO::Fitting model to feature number 55, COA.PWY..coenzyme.A.biosynthesis.I
## 2025-02-26 12:08:38.790685 INFO::Fitting model to feature number 56, PWY.4242..pantothenate.and.coenzyme.A.biosynthesis.III
## 2025-02-26 12:08:38.793031 INFO::Fitting model to feature number 57, PWY.6703..preQ0.biosynthesis
## 2025-02-26 12:08:38.795388 INFO::Fitting model to feature number 58, THISYN.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.I
## 2025-02-26 12:08:38.797764 INFO::Fitting model to feature number 59, HISDEG.PWY..L.histidine.degradation.I
## 2025-02-26 12:08:38.800092 INFO::Fitting model to feature number 60, PWY.5659..GDP.mannose.biosynthesis
## 2025-02-26 12:08:38.802396 INFO::Fitting model to feature number 61, PWY.5030..L.histidine.degradation.III
## 2025-02-26 12:08:38.804957 INFO::Fitting model to feature number 62, PWY.5484..glycolysis.II..from.fructose.6.phosphate.
## 2025-02-26 12:08:38.80728 INFO::Fitting model to feature number 63, PWY.3001..superpathway.of.L.isoleucine.biosynthesis.I
## 2025-02-26 12:08:38.809581 INFO::Fitting model to feature number 64, PWY.6125..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.II
## 2025-02-26 12:08:38.811944 INFO::Fitting model to feature number 65, TRPSYN.PWY..L.tryptophan.biosynthesis
## 2025-02-26 12:08:38.814245 INFO::Fitting model to feature number 66, GLYCOLYSIS..glycolysis.I..from.glucose.6.phosphate.
## 2025-02-26 12:08:38.816557 INFO::Fitting model to feature number 67, PWY.724..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.II
## 2025-02-26 12:08:38.818898 INFO::Fitting model to feature number 68, PWY.6897..thiamin.salvage.II
## 2025-02-26 12:08:38.821231 INFO::Fitting model to feature number 69, BRANCHED.CHAIN.AA.SYN.PWY..superpathway.of.branched.amino.acid.biosynthesis
## 2025-02-26 12:08:38.823586 INFO::Fitting model to feature number 70, CALVIN.PWY..Calvin.Benson.Bassham.cycle
## 2025-02-26 12:08:38.825945 INFO::Fitting model to feature number 71, PWY.841..superpathway.of.purine.nucleotides.de.novo.biosynthesis.I
## 2025-02-26 12:08:38.828324 INFO::Fitting model to feature number 72, COMPLETE.ARO.PWY..superpathway.of.aromatic.amino.acid.biosynthesis
## 2025-02-26 12:08:38.830711 INFO::Fitting model to feature number 73, PWY.7220..adenosine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2025-02-26 12:08:38.833059 INFO::Fitting model to feature number 74, PWY.7222..guanosine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2025-02-26 12:08:38.835387 INFO::Fitting model to feature number 75, DENOVOPURINE2.PWY..superpathway.of.purine.nucleotides.de.novo.biosynthesis.II
## 2025-02-26 12:08:38.837705 INFO::Fitting model to feature number 76, PWY.7357..thiamin.formation.from.pyrithiamine.and.oxythiamine..yeast.
## 2025-02-26 12:08:38.840057 INFO::Fitting model to feature number 77, PWY.5103..L.isoleucine.biosynthesis.III
## 2025-02-26 12:08:38.842365 INFO::Fitting model to feature number 78, ARO.PWY..chorismate.biosynthesis.I
## 2025-02-26 12:08:38.844738 INFO::Fitting model to feature number 79, PWY.7282..4.amino.2.methyl.5.phosphomethylpyrimidine.biosynthesis..yeast.
## 2025-02-26 12:08:38.847052 INFO::Fitting model to feature number 80, PWY0.845..superpathway.of.pyridoxal.5..phosphate.biosynthesis.and.salvage
## 2025-02-26 12:08:38.849358 INFO::Fitting model to feature number 81, ANAEROFRUCAT.PWY..homolactic.fermentation
## 2025-02-26 12:08:38.851697 INFO::Fitting model to feature number 82, PWY.6892..thiazole.biosynthesis.I..E..coli.
## 2025-02-26 12:08:38.854048 INFO::Fitting model to feature number 83, RHAMCAT.PWY..L.rhamnose.degradation.I
## 2025-02-26 12:08:38.856369 INFO::Fitting model to feature number 84, PYRIDOXSYN.PWY..pyridoxal.5..phosphate.biosynthesis.I
## 2025-02-26 12:08:38.858695 INFO::Fitting model to feature number 85, PWY.1269..CMP.3.deoxy.D.manno.octulosonate.biosynthesis.I
## 2025-02-26 12:08:38.861025 INFO::Fitting model to feature number 86, PHOSLIPSYN.PWY..superpathway.of.phospholipid.biosynthesis.I..bacteria.
## 2025-02-26 12:08:38.863346 INFO::Fitting model to feature number 87, CITRULBIO.PWY..L.citrulline.biosynthesis
## 2025-02-26 12:08:38.865654 INFO::Fitting model to feature number 88, NAGLIPASYN.PWY..lipid.IVA.biosynthesis
## 2025-02-26 12:08:38.868006 INFO::Fitting model to feature number 89, BIOTIN.BIOSYNTHESIS.PWY..biotin.biosynthesis.I
## 2025-02-26 12:08:38.87033 INFO::Fitting model to feature number 90, PWY.5154..L.arginine.biosynthesis.III..via.N.acetyl.L.citrulline.
## 2025-02-26 12:08:38.872647 INFO::Fitting model to feature number 91, FASYN.ELONG.PWY..fatty.acid.elongation....saturated
## 2025-02-26 12:08:38.875013 INFO::Fitting model to feature number 92, PENTOSE.P.PWY..pentose.phosphate.pathway
## 2025-02-26 12:08:38.877378 INFO::Fitting model to feature number 93, PWYG.321..mycolate.biosynthesis
## 2025-02-26 12:08:38.879692 INFO::Fitting model to feature number 94, PWY.6519..8.amino.7.oxononanoate.biosynthesis.I
## 2025-02-26 12:08:38.882074 INFO::Fitting model to feature number 95, PWY.7664..oleate.biosynthesis.IV..anaerobic.
## 2025-02-26 12:08:38.884392 INFO::Fitting model to feature number 96, PWY.4981..L.proline.biosynthesis.II..from.arginine.
## 2025-02-26 12:08:38.886709 INFO::Fitting model to feature number 97, GLYCOGENSYNTH.PWY..glycogen.biosynthesis.I..from.ADP.D.Glucose.
## 2025-02-26 12:08:38.889062 INFO::Fitting model to feature number 98, PWY.7388..octanoyl..acyl.carrier.protein..biosynthesis..mitochondria..yeast.
## 2025-02-26 12:08:38.891587 INFO::Fitting model to feature number 99, NONOXIPENT.PWY..pentose.phosphate.pathway..non.oxidative.branch.
## 2025-02-26 12:08:38.893947 INFO::Fitting model to feature number 100, COBALSYN.PWY..adenosylcobalamin.salvage.from.cobinamide.I
## 2025-02-26 12:08:38.896294 INFO::Fitting model to feature number 101, PWY.5989..stearate.biosynthesis.II..bacteria.and.plants.
## 2025-02-26 12:08:38.898631 INFO::Fitting model to feature number 102, PWY.6282..palmitoleate.biosynthesis.I..from..5Z..dodec.5.enoate.
## 2025-02-26 12:08:38.90098 INFO::Fitting model to feature number 103, PWY0.862...5Z..dodec.5.enoate.biosynthesis
## 2025-02-26 12:08:38.903303 INFO::Fitting model to feature number 104, PWY.7400..L.arginine.biosynthesis.IV..archaebacteria.
## 2025-02-26 12:08:38.905642 INFO::Fitting model to feature number 105, ARGSYN.PWY..L.arginine.biosynthesis.I..via.L.ornithine.
## 2025-02-26 12:08:38.907972 INFO::Fitting model to feature number 106, PWY.4984..urea.cycle
## 2025-02-26 12:08:38.910373 INFO::Fitting model to feature number 107, FASYN.INITIAL.PWY..superpathway.of.fatty.acid.biosynthesis.initiation..E..coli.
## 2025-02-26 12:08:38.912753 INFO::Fitting model to feature number 108, PWY4FS.7..phosphatidylglycerol.biosynthesis.I..plastidic.
## 2025-02-26 12:08:38.9151 INFO::Fitting model to feature number 109, PWY4FS.8..phosphatidylglycerol.biosynthesis.II..non.plastidic.
## 2025-02-26 12:08:38.917427 INFO::Fitting model to feature number 110, ARGSYNBSUB.PWY..L.arginine.biosynthesis.II..acetyl.cycle.
## 2025-02-26 12:08:38.919766 INFO::Fitting model to feature number 111, PWY.6317..galactose.degradation.I..Leloir.pathway.
## 2025-02-26 12:08:38.922136 INFO::Fitting model to feature number 112, PWY66.422..D.galactose.degradation.V..Leloir.pathway.
## 2025-02-26 12:08:38.924482 INFO::Fitting model to feature number 113, GLUTORN.PWY..L.ornithine.biosynthesis
## 2025-02-26 12:08:38.926825 INFO::Fitting model to feature number 114, PWY.2941..L.lysine.biosynthesis.II
## 2025-02-26 12:08:38.929137 INFO::Fitting model to feature number 115, PWY0.1296..purine.ribonucleosides.degradation
## 2025-02-26 12:08:38.931447 INFO::Fitting model to feature number 116, NAD.BIOSYNTHESIS.II..NAD.salvage.pathway.II
## 2025-02-26 12:08:38.933791 INFO::Fitting model to feature number 117, HSERMETANA.PWY..L.methionine.biosynthesis.III
## 2025-02-26 12:08:38.936152 INFO::Fitting model to feature number 118, PWY0.162..superpathway.of.pyrimidine.ribonucleotides.de.novo.biosynthesis
## 2025-02-26 12:08:38.938446 INFO::Fitting model to feature number 119, PWY.7208..superpathway.of.pyrimidine.nucleobases.salvage
## 2025-02-26 12:08:38.94078 INFO::Fitting model to feature number 120, TEICHOICACID.PWY..teichoic.acid..poly.glycerol..biosynthesis
## 2025-02-26 12:08:38.943092 INFO::Fitting model to feature number 121, PWY.6305..putrescine.biosynthesis.IV
## 2025-02-26 12:08:38.945453 INFO::Fitting model to feature number 122, GLUCONEO.PWY..gluconeogenesis.I
## 2025-02-26 12:08:38.947865 INFO::Fitting model to feature number 123, PWY.621..sucrose.degradation.III..sucrose.invertase.
## 2025-02-26 12:08:38.950218 INFO::Fitting model to feature number 124, PWY.6901..superpathway.of.glucose.and.xylose.degradation
## 2025-02-26 12:08:38.952569 INFO::Fitting model to feature number 125, POLYISOPRENSYN.PWY..polyisoprenoid.biosynthesis..E..coli.
## 2025-02-26 12:08:38.954914 INFO::Fitting model to feature number 126, PWY.7323..superpathway.of.GDP.mannose.derived.O.antigen.building.blocks.biosynthesis
## 2025-02-26 12:08:38.957233 INFO::Fitting model to feature number 127, PWY.5941..glycogen.degradation.II..eukaryotic.
## 2025-02-26 12:08:38.959559 INFO::Fitting model to feature number 128, PWY.6527..stachyose.degradation
## 2025-02-26 12:08:38.961884 INFO::Fitting model to feature number 129, PWY0.1261..anhydromuropeptides.recycling
## 2025-02-26 12:08:38.964209 INFO::Fitting model to feature number 130, PWY66.400..glycolysis.VI..metazoan.
## 2025-02-26 12:08:38.966526 INFO::Fitting model to feature number 131, PWY0.166..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis..E..coli.
## 2025-02-26 12:08:38.968941 INFO::Fitting model to feature number 132, GLYCOLYSIS.E.D..superpathway.of.glycolysis.and.Entner.Doudoroff
## 2025-02-26 12:08:38.971274 INFO::Fitting model to feature number 133, PWY.7187..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2025-02-26 12:08:38.973609 INFO::Fitting model to feature number 134, PWY.7456..mannan.degradation
## 2025-02-26 12:08:38.976031 INFO::Fitting model to feature number 135, PWY.7184..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.I
## 2025-02-26 12:08:38.97842 INFO::Fitting model to feature number 136, OANTIGEN.PWY..O.antigen.building.blocks.biosynthesis..E..coli.
## 2025-02-26 12:08:38.98083 INFO::Fitting model to feature number 137, COLANSYN.PWY..colanic.acid.building.blocks.biosynthesis
## 2025-02-26 12:08:38.983177 INFO::Fitting model to feature number 138, PWY.6545..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.III
## 2025-02-26 12:08:38.985579 INFO::Fitting model to feature number 139, PWY.6895..superpathway.of.thiamin.diphosphate.biosynthesis.II
## 2025-02-26 12:08:38.987984 INFO::Fitting model to feature number 140, PWY.6608..guanosine.nucleotides.degradation.III
## 2025-02-26 12:08:38.99031 INFO::Fitting model to feature number 141, TCA..TCA.cycle.I..prokaryotic.
## 2025-02-26 12:08:38.992646 INFO::Fitting model to feature number 142, PWY.6859..all.trans.farnesol.biosynthesis
## 2025-02-26 12:08:38.995025 INFO::Fitting model to feature number 143, PWY.5100..pyruvate.fermentation.to.acetate.and.lactate.II
## 2025-02-26 12:08:38.997354 INFO::Fitting model to feature number 144, PWY.6572..chondroitin.sulfate.degradation.I..bacterial.
## 2025-02-26 12:08:38.9997 INFO::Fitting model to feature number 145, PWY.6471..peptidoglycan.biosynthesis.IV..Enterococcus.faecium.
## 2025-02-26 12:08:39.002082 INFO::Fitting model to feature number 146, PWY.6470..peptidoglycan.biosynthesis.V...beta..lactam.resistance.
## 2025-02-26 12:08:39.004426 INFO::Fitting model to feature number 147, PWY.5188..tetrapyrrole.biosynthesis.I..from.glutamate.
## 2025-02-26 12:08:39.006797 INFO::Fitting model to feature number 148, GLUCUROCAT.PWY..superpathway.of..beta..D.glucuronide.and.D.glucuronate.degradation
## 2025-02-26 12:08:39.009138 INFO::Fitting model to feature number 149, PWY.6749..CMP.legionaminate.biosynthesis.I
## 2025-02-26 12:08:39.011523 INFO::Fitting model to feature number 150, P164.PWY..purine.nucleobases.degradation.I..anaerobic.
## 2025-02-26 12:08:39.013891 INFO::Fitting model to feature number 151, PWY.7198..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.IV
## 2025-02-26 12:08:39.016346 INFO::Fitting model to feature number 152, PWY.7211..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis
## 2025-02-26 12:08:39.018669 INFO::Fitting model to feature number 153, PWY66.409..superpathway.of.purine.nucleotide.salvage
## 2025-02-26 12:08:39.021041 INFO::Fitting model to feature number 154, GALACT.GLUCUROCAT.PWY..superpathway.of.hexuronide.and.hexuronate.degradation
## 2025-02-26 12:08:39.023389 INFO::Fitting model to feature number 155, UDPNAGSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.I
## 2025-02-26 12:08:39.025808 INFO::Fitting model to feature number 156, PWY.6353..purine.nucleotides.degradation.II..aerobic.
## 2025-02-26 12:08:39.028929 INFO::Fitting model to feature number 157, PWY.7197..pyrimidine.deoxyribonucleotide.phosphorylation
## 2025-02-26 12:08:39.031332 INFO::Fitting model to feature number 158, PWY.7242..D.fructuronate.degradation
## 2025-02-26 12:08:39.033674 INFO::Fitting model to feature number 159, PWY.7196..superpathway.of.pyrimidine.ribonucleosides.salvage
## 2025-02-26 12:08:39.036062 INFO::Fitting model to feature number 160, PWY.5690..TCA.cycle.II..plants.and.fungi.
## 2025-02-26 12:08:39.038379 INFO::Fitting model to feature number 161, PWY.5177..glutaryl.CoA.degradation
## 2025-02-26 12:08:39.040717 INFO::Fitting model to feature number 162, PWY.6478..GDP.D.glycero..alpha..D.manno.heptose.biosynthesis
## 2025-02-26 12:08:39.043065 INFO::Fitting model to feature number 163, HEXITOLDEGSUPER.PWY..superpathway.of.hexitol.degradation..bacteria.
## 2025-02-26 12:08:39.045404 INFO::Fitting model to feature number 164, GALACTUROCAT.PWY..D.galacturonate.degradation.I
## 2025-02-26 12:08:39.047778 INFO::Fitting model to feature number 165, PWY.5104..L.isoleucine.biosynthesis.IV
## 2025-02-26 12:08:39.050098 INFO::Fitting model to feature number 166, PWY.5505..L.glutamate.and.L.glutamine.biosynthesis
## 2025-02-26 12:08:39.052423 INFO::Fitting model to feature number 167, P441.PWY..superpathway.of.N.acetylneuraminate.degradation
## 2025-02-26 12:08:39.054795 INFO::Fitting model to feature number 168, PWY.6606..guanosine.nucleotides.degradation.II
## 2025-02-26 12:08:39.057145 INFO::Fitting model to feature number 169, SALVADEHYPOX.PWY..adenosine.nucleotides.degradation.II
## 2025-02-26 12:08:39.059503 INFO::Fitting model to feature number 170, PWY.5005..biotin.biosynthesis.II
## 2025-02-26 12:08:39.061908 INFO::Fitting model to feature number 171, PWY.6549..L.glutamine.biosynthesis.III
## 2025-02-26 12:08:39.064238 INFO::Fitting model to feature number 172, GLCMANNANAUT.PWY..superpathway.of.N.acetylglucosamine..N.acetylmannosamine.and.N.acetylneuraminate.degradation
## 2025-02-26 12:08:39.066576 INFO::Fitting model to feature number 173, PWY0.1241..ADP.L.glycero..beta..D.manno.heptose.biosynthesis
## 2025-02-26 12:08:39.068957 INFO::Fitting model to feature number 174, PWY.6507..4.deoxy.L.threo.hex.4.enopyranuronate.degradation
## 2025-02-26 12:08:39.071293 INFO::Fitting model to feature number 175, GALACTARDEG.PWY..D.galactarate.degradation.I
## 2025-02-26 12:08:39.073625 INFO::Fitting model to feature number 176, GLUCARGALACTSUPER.PWY..superpathway.of.D.glucarate.and.D.galactarate.degradation
## 2025-02-26 12:08:39.076152 INFO::Fitting model to feature number 177, PWY.5676..acetyl.CoA.fermentation.to.butanoate.II
## 2025-02-26 12:08:39.078666 INFO::Fitting model to feature number 178, PWY.5347..superpathway.of.L.methionine.biosynthesis..transsulfuration.
## 2025-02-26 12:08:39.081064 INFO::Fitting model to feature number 179, PWY.5138..unsaturated..even.numbered.fatty.acid..beta..oxidation
## 2025-02-26 12:08:39.08341 INFO::Fitting model to feature number 180, MET.SAM.PWY..superpathway.of.S.adenosyl.L.methionine.biosynthesis
## 2025-02-26 12:08:39.085794 INFO::Fitting model to feature number 181, PWY66.399..gluconeogenesis.III
## 2025-02-26 12:08:39.088166 INFO::Fitting model to feature number 182, PWY0.1297..superpathway.of.purine.deoxyribonucleosides.degradation
## 2025-02-26 12:08:39.090527 INFO::Fitting model to feature number 183, METSYN.PWY..L.homoserine.and.L.methionine.biosynthesis
## 2025-02-26 12:08:39.093005 INFO::Fitting model to feature number 184, PWY.7332..superpathway.of.UDP.N.acetylglucosamine.derived.O.antigen.building.blocks.biosynthesis
## 2025-02-26 12:08:39.095369 INFO::Fitting model to feature number 185, PWY.6588..pyruvate.fermentation.to.acetone
## 2025-02-26 12:08:39.097793 INFO::Fitting model to feature number 186, PWY.6891..thiazole.biosynthesis.II..Bacillus.
## 2025-02-26 12:08:39.100284 INFO::Fitting model to feature number 187, GLUCARDEG.PWY..D.glucarate.degradation.I
## 2025-02-26 12:08:39.102671 INFO::Fitting model to feature number 188, METH.ACETATE.PWY..methanogenesis.from.acetate
## 2025-02-26 12:08:39.105068 INFO::Fitting model to feature number 189, PWY.7013..L.1.2.propanediol.degradation
## 2025-02-26 12:08:39.107445 INFO::Fitting model to feature number 190, PWY.6590..superpathway.of.Clostridium.acetobutylicum.acidogenic.fermentation
## 2025-02-26 12:08:39.109867 INFO::Fitting model to feature number 191, FAO.PWY..fatty.acid..beta..oxidation.I
## 2025-02-26 12:08:39.112214 INFO::Fitting model to feature number 192, PWY.6318..L.phenylalanine.degradation.IV..mammalian..via.side.chain.
## 2025-02-26 12:08:39.114576 INFO::Fitting model to feature number 193, CENTFERM.PWY..pyruvate.fermentation.to.butanoate
## 2025-02-26 12:08:39.116967 INFO::Fitting model to feature number 194, GOLPDLCAT.PWY..superpathway.of.glycerol.degradation.to.1.3.propanediol
## 2025-02-26 12:08:39.119337 INFO::Fitting model to feature number 195, PWY.5136..fatty.acid..beta..oxidation.II..peroxisome.
## 2025-02-26 12:08:39.121699 INFO::Fitting model to feature number 196, PWY66.391..fatty.acid..beta..oxidation.VI..peroxisome.
## 2025-02-26 12:08:39.124102 INFO::Fitting model to feature number 197, HOMOSER.METSYN.PWY..L.methionine.biosynthesis.I
## 2025-02-26 12:08:39.126468 INFO::Fitting model to feature number 198, METHGLYUT.PWY..superpathway.of.methylglyoxal.degradation
## 2025-02-26 12:08:39.128875 INFO::Fitting model to feature number 199, PWY.7288..fatty.acid..beta..oxidation..peroxisome..yeast.
## 2025-02-26 12:08:39.131235 INFO::Fitting model to feature number 200, PWY.7383..anaerobic.energy.metabolism..invertebrates..cytosol.
## 2025-02-26 12:08:39.133611 INFO::Fitting model to feature number 201, P162.PWY..L.glutamate.degradation.V..via.hydroxyglutarate.
## 2025-02-26 12:08:39.136008 INFO::Fitting model to feature number 202, PWY.4041...gamma..glutamyl.cycle
## 2025-02-26 12:08:39.138369 INFO::Fitting model to feature number 203, P108.PWY..pyruvate.fermentation.to.propanoate.I
## 2025-02-26 12:08:39.140805 INFO::Fitting model to feature number 204, PWY.5022..4.aminobutanoate.degradation.V
## 2025-02-26 12:08:39.143193 INFO::Fitting model to feature number 205, GLUDEG.I.PWY..GABA.shunt
## 2025-02-26 12:08:39.145571 INFO::Fitting model to feature number 206, PWY.5677..succinate.fermentation.to.butanoate
## 2025-02-26 12:08:39.147963 INFO::Fitting model to feature number 207, P163.PWY..L.lysine.fermentation.to.acetate.and.butanoate
## 2025-02-26 12:08:39.150335 INFO::Fitting model to feature number 208, PWY.3781..aerobic.respiration.I..cytochrome.c.
## 2025-02-26 12:08:39.152704 INFO::Fitting model to feature number 209, PWY.5083..NAD.NADH.phosphorylation.and.dephosphorylation
## 2025-02-26 12:08:39.15513 INFO::Fitting model to feature number 210, DAPLYSINESYN.PWY..L.lysine.biosynthesis.I
## 2025-02-26 12:08:39.157499 INFO::Fitting model to feature number 211, P461.PWY..hexitol.fermentation.to.lactate..formate..ethanol.and.acetate
## 2025-02-26 12:08:39.159902 INFO::Fitting model to feature number 212, PWY.1861..formaldehyde.assimilation.II..RuMP.Cycle.
## 2025-02-26 12:08:39.162253 INFO::Fitting model to feature number 213, PWY.7312..dTDP.D..beta..fucofuranose.biosynthesis
## 2025-02-26 12:08:39.16477 INFO::Fitting model to feature number 214, PWY.6630..superpathway.of.L.tyrosine.biosynthesis
## 2025-02-26 12:08:39.167717 INFO::Fitting model to feature number 215, PWY.6595..superpathway.of.guanosine.nucleotides.degradation..plants.
## 2025-02-26 12:08:39.170683 INFO::Fitting model to feature number 216, ARG.POLYAMINE.SYN..superpathway.of.arginine.and.polyamine.biosynthesis
## 2025-02-26 12:08:39.173681 INFO::Fitting model to feature number 217, PWY.7003..glycerol.degradation.to.butanol
## 2025-02-26 12:08:39.176823 INFO::Fitting model to feature number 218, P185.PWY..formaldehyde.assimilation.III..dihydroxyacetone.cycle.
## 2025-02-26 12:08:39.179777 INFO::Fitting model to feature number 219, POLYAMSYN.PWY..superpathway.of.polyamine.biosynthesis.I
## 2025-02-26 12:08:39.182694 INFO::Fitting model to feature number 220, PWY0.1298..superpathway.of.pyrimidine.deoxyribonucleosides.degradation
## 2025-02-26 12:08:39.185652 INFO::Fitting model to feature number 221, LACTOSECAT.PWY..lactose.and.galactose.degradation.I
## 2025-02-26 12:08:39.188613 INFO::Fitting model to feature number 222, PWY4LZ.257..superpathway.of.fermentation..Chlamydomonas.reinhardtii.
## 2025-02-26 12:08:39.191572 INFO::Fitting model to feature number 223, P161.PWY..acetylene.degradation
## 2025-02-26 12:08:39.194521 INFO::Fitting model to feature number 224, RUMP.PWY..formaldehyde.oxidation.I
## 2025-02-26 12:08:39.197491 INFO::Fitting model to feature number 225, PPGPPMET.PWY..ppGpp.biosynthesis
## 2025-02-26 12:08:39.200511 INFO::Fitting model to feature number 226, PWY.5304..superpathway.of.sulfur.oxidation..Acidianus.ambivalens.
## 2025-02-26 12:08:39.203533 INFO::Fitting model to feature number 227, PWY.6531..mannitol.cycle
## 2025-02-26 12:08:39.206523 INFO::Fitting model to feature number 228, FUC.RHAMCAT.PWY..superpathway.of.fucose.and.rhamnose.degradation
## 2025-02-26 12:08:39.209492 INFO::Fitting model to feature number 229, PWY.7315..dTDP.N.acetylthomosamine.biosynthesis
## 2025-02-26 12:08:39.212434 INFO::Fitting model to feature number 230, FUCCAT.PWY..fucose.degradation
## 2025-02-26 12:08:39.215403 INFO::Fitting model to feature number 231, PWY.7210..pyrimidine.deoxyribonucleotides.biosynthesis.from.CTP
## 2025-02-26 12:08:39.218408 INFO::Fitting model to feature number 232, FOLSYN.PWY..superpathway.of.tetrahydrofolate.biosynthesis.and.salvage
## 2025-02-26 12:08:39.221431 INFO::Fitting model to feature number 233, PWY.6612..superpathway.of.tetrahydrofolate.biosynthesis
## 2025-02-26 12:08:39.224392 INFO::Fitting model to feature number 234, PWY0.1061..superpathway.of.L.alanine.biosynthesis
## 2025-02-26 12:08:39.227481 INFO::Fitting model to feature number 235, PWY.5189..tetrapyrrole.biosynthesis.II..from.glycine.
## 2025-02-26 12:08:39.230446 INFO::Fitting model to feature number 236, PWY.6876..isopropanol.biosynthesis
## 2025-02-26 12:08:39.233417 INFO::Fitting model to feature number 237, PWY0.781..aspartate.superpathway
## 2025-02-26 12:08:39.236394 INFO::Fitting model to feature number 238, P4.PWY..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.I
## 2025-02-26 12:08:39.239383 INFO::Fitting model to feature number 239, PWY0.1479..tRNA.processing
## 2025-02-26 12:08:39.242375 INFO::Fitting model to feature number 240, GLYCOLYSIS.TCA.GLYOX.BYPASS..superpathway.of.glycolysis..pyruvate.dehydrogenase..TCA..and.glyoxylate.bypass
## 2025-02-26 12:08:39.245419 INFO::Fitting model to feature number 241, PYRIDNUCSAL.PWY..NAD.salvage.pathway.I
## 2025-02-26 12:08:39.248418 INFO::Fitting model to feature number 242, GLYCOCAT.PWY..glycogen.degradation.I..bacterial.
## 2025-02-26 12:08:39.251421 INFO::Fitting model to feature number 243, PWY.5723..Rubisco.shunt
## 2025-02-26 12:08:39.254396 INFO::Fitting model to feature number 244, TCA.GLYOX.BYPASS..superpathway.of.glyoxylate.bypass.and.TCA
## 2025-02-26 12:08:39.257376 INFO::Fitting model to feature number 245, PWY.7385..1.3.propanediol.biosynthesis..engineered.
## 2025-02-26 12:08:39.260372 INFO::Fitting model to feature number 246, PWY.7254..TCA.cycle.VII..acetate.producers.
## 2025-02-26 12:08:39.263333 INFO::Fitting model to feature number 247, GLYOXYLATE.BYPASS..glyoxylate.cycle
## 2025-02-26 12:08:39.266336 INFO::Fitting model to feature number 248, PWY.6731..starch.degradation.III
## 2025-02-26 12:08:39.269367 INFO::Fitting model to feature number 249, PWY.7328..superpathway.of.UDP.glucose.derived.O.antigen.building.blocks.biosynthesis
## 2025-02-26 12:08:39.272346 INFO::Fitting model to feature number 250, POLYAMINSYN3.PWY..superpathway.of.polyamine.biosynthesis.II
## 2025-02-26 12:08:39.275319 INFO::Fitting model to feature number 251, PWY.2723..trehalose.degradation.V
## 2025-02-26 12:08:39.278306 INFO::Fitting model to feature number 252, GLUCOSE1PMETAB.PWY..glucose.and.glucose.1.phosphate.degradation
## 2025-02-26 12:08:39.281312 INFO::Fitting model to feature number 253, PWY.5384..sucrose.degradation.IV..sucrose.phosphorylase.
## 2025-02-26 12:08:39.28431 INFO::Fitting model to feature number 254, PWY.7046..4.coumarate.degradation..anaerobic.
## 2025-02-26 12:08:39.287294 INFO::Fitting model to feature number 255, PWY66.389..phytol.degradation
## 2025-02-26 12:08:39.290292 INFO::Fitting model to feature number 256, PWY.5971..palmitate.biosynthesis.II..bacteria.and.plants.
## 2025-02-26 12:08:39.2933 INFO::Fitting model to feature number 257, PWY.6629..superpathway.of.L.tryptophan.biosynthesis
## 2025-02-26 12:08:39.296268 INFO::Fitting model to feature number 258, PWY.5345..superpathway.of.L.methionine.biosynthesis..by.sulfhydrylation.
## 2025-02-26 12:08:39.299242 INFO::Fitting model to feature number 259, SULFATE.CYS.PWY..superpathway.of.sulfate.assimilation.and.cysteine.biosynthesis
## 2025-02-26 12:08:39.302218 INFO::Fitting model to feature number 260, PWY.5838..superpathway.of.menaquinol.8.biosynthesis.I
## 2025-02-26 12:08:39.305242 INFO::Fitting model to feature number 261, PRPP.PWY..superpathway.of.histidine..purine..and.pyrimidine.biosynthesis
## 2025-02-26 12:08:39.308275 INFO::Fitting model to feature number 262, PWY.5845..superpathway.of.menaquinol.9.biosynthesis
## 2025-02-26 12:08:39.311285 INFO::Fitting model to feature number 263, PWY.6113..superpathway.of.mycolate.biosynthesis
## 2025-02-26 12:08:39.31427 INFO::Fitting model to feature number 264, PWY.5897..superpathway.of.menaquinol.11.biosynthesis
## 2025-02-26 12:08:39.317223 INFO::Fitting model to feature number 265, PWY.5898..superpathway.of.menaquinol.12.biosynthesis
## 2025-02-26 12:08:39.320269 INFO::Fitting model to feature number 266, PWY.5899..superpathway.of.menaquinol.13.biosynthesis
## 2025-02-26 12:08:39.323352 INFO::Fitting model to feature number 267, PWY.7117..C4.photosynthetic.carbon.assimilation.cycle..PEPCK.type
## 2025-02-26 12:08:39.326371 INFO::Fitting model to feature number 268, PWY.6969..TCA.cycle.V..2.oxoglutarate.ferredoxin.oxidoreductase.
## 2025-02-26 12:08:39.32942 INFO::Fitting model to feature number 269, PWY.6628..superpathway.of.L.phenylalanine.biosynthesis
## 2025-02-26 12:08:39.332445 INFO::Fitting model to feature number 270, PWY.5861..superpathway.of.demethylmenaquinol.8.biosynthesis
## 2025-02-26 12:08:39.335445 INFO::Fitting model to feature number 271, PWY.5862..superpathway.of.demethylmenaquinol.9.biosynthesis
## 2025-02-26 12:08:39.338471 INFO::Fitting model to feature number 272, FERMENTATION.PWY..mixed.acid.fermentation
## 2025-02-26 12:08:39.341486 INFO::Fitting model to feature number 273, PWY.241..C4.photosynthetic.carbon.assimilation.cycle..NADP.ME.type
## 2025-02-26 12:08:39.344481 INFO::Fitting model to feature number 274, SO4ASSIM.PWY..sulfate.reduction.I..assimilatory.
## 2025-02-26 12:08:39.347483 INFO::Fitting model to feature number 275, KETOGLUCONMET.PWY..ketogluconate.metabolism
## 2025-02-26 12:08:39.3505 INFO::Fitting model to feature number 276, PWY.5840..superpathway.of.menaquinol.7.biosynthesis
## 2025-02-26 12:08:39.353529 INFO::Fitting model to feature number 277, PWY.5850..superpathway.of.menaquinol.6.biosynthesis.I
## 2025-02-26 12:08:39.356557 INFO::Fitting model to feature number 278, PWY.5896..superpathway.of.menaquinol.10.biosynthesis
## 2025-02-26 12:08:39.359565 INFO::Fitting model to feature number 279, P105.PWY..TCA.cycle.IV..2.oxoglutarate.decarboxylase.
## 2025-02-26 12:08:39.362568 INFO::Fitting model to feature number 280, PWY.5913..TCA.cycle.VI..obligate.autotrophs.
## 2025-02-26 12:08:39.3656 INFO::Fitting model to feature number 281, PWY.561..superpathway.of.glyoxylate.cycle.and.fatty.acid.degradation
## 2025-02-26 12:08:39.368582 INFO::Fitting model to feature number 282, PWY.4702..phytate.degradation.I
## 2025-02-26 12:08:39.371575 INFO::Fitting model to feature number 283, PWY.6803..phosphatidylcholine.acyl.editing
## 2025-02-26 12:08:39.374597 INFO::Fitting model to feature number 284, PWY.7115..C4.photosynthetic.carbon.assimilation.cycle..NAD.ME.type
## 2025-02-26 12:08:39.377631 INFO::Fitting model to feature number 285, PWY.5918..superpathay.of.heme.biosynthesis.from.glutamate
## 2025-02-26 12:08:39.380618 INFO::Fitting model to feature number 286, PWY.7234..inosine.5..phosphate.biosynthesis.III
## 2025-02-26 12:08:39.383638 INFO::Fitting model to feature number 287, PWY.6285..superpathway.of.fatty.acids.biosynthesis..E..coli.
## 2025-02-26 12:08:39.386647 INFO::Fitting model to feature number 288, PWY.5860..superpathway.of.demethylmenaquinol.6.biosynthesis.I
## 2025-02-26 12:08:39.389627 INFO::Fitting model to feature number 289, PWY.5863..superpathway.of.phylloquinol.biosynthesis
## 2025-02-26 12:08:39.3927 INFO::Fitting model to feature number 290, HEME.BIOSYNTHESIS.II..heme.biosynthesis.I..aerobic.
## 2025-02-26 12:08:39.39579 INFO::Fitting model to feature number 291, PWY.821..superpathway.of.sulfur.amino.acid.biosynthesis..Saccharomyces.cerevisiae.
## 2025-02-26 12:08:39.39879 INFO::Fitting model to feature number 292, PWY.5675..nitrate.reduction.V..assimilatory.
## 2025-02-26 12:08:39.401818 INFO::Fitting model to feature number 293, PWY.5791..1.4.dihydroxy.2.naphthoate.biosynthesis.II..plants.
## 2025-02-26 12:08:39.404808 INFO::Fitting model to feature number 294, PWY.5837..1.4.dihydroxy.2.naphthoate.biosynthesis.I
## 2025-02-26 12:08:39.407812 INFO::Fitting model to feature number 295, PWY.5920..superpathway.of.heme.biosynthesis.from.glycine
## 2025-02-26 12:08:39.410934 INFO::Fitting model to feature number 296, PWY.7204..pyridoxal.5..phosphate.salvage.II..plants.
## 2025-02-26 12:08:39.413929 INFO::Fitting model to feature number 297, ENTBACSYN.PWY..enterobactin.biosynthesis
## 2025-02-26 12:08:39.416988 INFO::Fitting model to feature number 298, PWY0.1415..superpathway.of.heme.biosynthesis.from.uroporphyrinogen.III
## 2025-02-26 12:08:39.419973 INFO::Fitting model to feature number 299, HEMESYN2.PWY..heme.biosynthesis.II..anaerobic.
## 2025-02-26 12:08:39.42298 INFO::Fitting model to feature number 300, PWY.6284..superpathway.of.unsaturated.fatty.acids.biosynthesis..E..coli.
## 2025-02-26 12:08:39.425992 INFO::Fitting model to feature number 301, P42.PWY..incomplete.reductive.TCA.cycle
## 2025-02-26 12:08:39.429101 INFO::Fitting model to feature number 302, PWY.5656..mannosylglycerate.biosynthesis.I
## 2025-02-26 12:08:39.432177 INFO::Fitting model to feature number 303, PWY.5173..superpathway.of.acetyl.CoA.biosynthesis
## 2025-02-26 12:08:39.435185 INFO::Fitting model to feature number 304, ECASYN.PWY..enterobacterial.common.antigen.biosynthesis
## 2025-02-26 12:08:39.438207 INFO::Fitting model to feature number 305, AST.PWY..L.arginine.degradation.II..AST.pathway.
## 2025-02-26 12:08:39.441243 INFO::Fitting model to feature number 306, PWY3O.355..stearate.biosynthesis.III..fungi.
## 2025-02-26 12:08:39.444211 INFO::Fitting model to feature number 307, PWY0.42..2.methylcitrate.cycle.I
## 2025-02-26 12:08:39.447187 INFO::Fitting model to feature number 308, PWY.7094..fatty.acid.salvage
## 2025-02-26 12:08:39.450206 INFO::Fitting model to feature number 309, PWY.5747..2.methylcitrate.cycle.II
## 2025-02-26 12:08:39.45321 INFO::Fitting model to feature number 310, PWY.5367..petroselinate.biosynthesis
## 2025-02-26 12:08:39.456257 INFO::Fitting model to feature number 311, PWY0.1533..methylphosphonate.degradation.I
## 2025-02-26 12:08:39.459341 INFO::Fitting model to feature number 312, PWY.6823..molybdenum.cofactor.biosynthesis
## 2025-02-26 12:08:39.462307 INFO::Fitting model to feature number 313, PWY.5705..allantoin.degradation.to.glyoxylate.III
## 2025-02-26 12:08:39.465335 INFO::Fitting model to feature number 314, LPSSYN.PWY..superpathway.of.lipopolysaccharide.biosynthesis
## 2025-02-26 12:08:39.468336 INFO::Fitting model to feature number 315, REDCITCYC..TCA.cycle.VIII..helicobacter.
## 2025-02-26 12:08:39.471325 INFO::Fitting model to feature number 316, GLYCOL.GLYOXDEG.PWY..superpathway.of.glycol.metabolism.and.degradation
## 2025-02-26 12:08:39.474323 INFO::Fitting model to feature number 317, P122.PWY..heterolactic.fermentation
## 2025-02-26 12:08:39.477431 INFO::Fitting model to feature number 318, KDO.NAGLIPASYN.PWY..superpathway.of..Kdo.2.lipid.A.biosynthesis
## 2025-02-26 12:08:39.480451 INFO::Fitting model to feature number 319, PWY.7446..sulfoglycolysis
## 2025-02-26 12:08:39.483465 INFO::Fitting model to feature number 320, PWY490.3..nitrate.reduction.VI..assimilatory.
## 2025-02-26 12:08:39.486557 INFO::Fitting model to feature number 321, PWY.7209..superpathway.of.pyrimidine.ribonucleosides.degradation
## 2025-02-26 12:08:39.489611 INFO::Fitting model to feature number 322, UBISYN.PWY..superpathway.of.ubiquinol.8.biosynthesis..prokaryotic.
## 2025-02-26 12:08:39.492631 INFO::Fitting model to feature number 323, PWY0.41..allantoin.degradation.IV..anaerobic.
## 2025-02-26 12:08:39.495639 INFO::Fitting model to feature number 324, PWY.5855..ubiquinol.7.biosynthesis..prokaryotic.
## 2025-02-26 12:08:39.49864 INFO::Fitting model to feature number 325, PWY.5856..ubiquinol.9.biosynthesis..prokaryotic.
## 2025-02-26 12:08:39.501678 INFO::Fitting model to feature number 326, PWY.5857..ubiquinol.10.biosynthesis..prokaryotic.
## 2025-02-26 12:08:39.504674 INFO::Fitting model to feature number 327, PWY.6708..ubiquinol.8.biosynthesis..prokaryotic.
## 2025-02-26 12:08:39.507674 INFO::Fitting model to feature number 328, PWY.5692..allantoin.degradation.to.glyoxylate.II
## 2025-02-26 12:08:39.54055 INFO::Fitting model to feature number 329, URDEGR.PWY..superpathway.of.allantoin.degradation.in.plants
## 2025-02-26 12:08:39.543261 INFO::Fitting model to feature number 330, PWY0.1338..polymyxin.resistance
## 2025-02-26 12:08:39.54561 INFO::Fitting model to feature number 331, P221.PWY..octane.oxidation
## 2025-02-26 12:08:39.547971 INFO::Fitting model to feature number 332, THREOCAT.PWY..superpathway.of.L.threonine.metabolism
## 2025-02-26 12:08:39.550321 INFO::Fitting model to feature number 333, NONMEVIPP.PWY..methylerythritol.phosphate.pathway.I
## 2025-02-26 12:08:39.55266 INFO::Fitting model to feature number 334, PWY.7560..methylerythritol.phosphate.pathway.II
## 2025-02-26 12:08:39.555005 INFO::Fitting model to feature number 335, PWY.5121..superpathway.of.geranylgeranyl.diphosphate.biosynthesis.II..via.MEP.
## 2025-02-26 12:08:39.557349 INFO::Fitting model to feature number 336, PWY.7392..taxadiene.biosynthesis..engineered.
## 2025-02-26 12:08:39.559685 INFO::Fitting model to feature number 337, PWY.6143..CMP.pseudaminate.biosynthesis
## 2025-02-26 12:08:39.562034 INFO::Fitting model to feature number 338, PWY.6270..isoprene.biosynthesis.I
## 2025-02-26 12:08:39.564376 INFO::Fitting model to feature number 339, ALLANTOINDEG.PWY..superpathway.of.allantoin.degradation.in.yeast
## 2025-02-26 12:08:39.566807 INFO::Fitting model to feature number 340, PWY.6263..superpathway.of.menaquinol.8.biosynthesis.II
## 2025-02-26 12:08:39.569155 INFO::Fitting model to feature number 341, PWY.7371..1.4.dihydroxy.6.naphthoate.biosynthesis.II
## 2025-02-26 12:08:39.571496 INFO::Fitting model to feature number 342, ARGORNPROST.PWY..arginine..ornithine.and.proline.interconversion
## 2025-02-26 12:08:39.573891 INFO::Fitting model to feature number 343, PWY66.398..TCA.cycle.III..animals.
## 2025-02-26 12:08:39.576247 INFO::Fitting model to feature number 344, PWY.5464..superpathway.of.cytosolic.glycolysis..plants...pyruvate.dehydrogenase.and.TCA.cycle
## 2025-02-26 12:08:39.578628 INFO::Fitting model to feature number 345, ORNDEG.PWY..superpathway.of.ornithine.degradation
## 2025-02-26 12:08:39.580994 INFO::Fitting model to feature number 346, P23.PWY..reductive.TCA.cycle.I
## 2025-02-26 12:08:39.583326 INFO::Fitting model to feature number 347, PWY0.1277..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation
## 2025-02-26 12:08:39.58566 INFO::Fitting model to feature number 348, ARGDEG.PWY..superpathway.of.L.arginine..putrescine..and.4.aminobutanoate.degradation
## 2025-02-26 12:08:39.588028 INFO::Fitting model to feature number 349, ORNARGDEG.PWY..superpathway.of.L.arginine.and.L.ornithine.degradation
## 2025-02-26 12:08:39.590374 INFO::Fitting model to feature number 350, HCAMHPDEG.PWY..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation.to.2.oxopent.4.enoate
## 2025-02-26 12:08:39.592763 INFO::Fitting model to feature number 351, PWY.6690..cinnamate.and.3.hydroxycinnamate.degradation.to.2.oxopent.4.enoate
## 2025-02-26 12:08:39.595121 INFO::Fitting model to feature number 352, PROTOCATECHUATE.ORTHO.CLEAVAGE.PWY..protocatechuate.degradation.II..ortho.cleavage.pathway.
## 2025-02-26 12:08:39.597462 INFO::Fitting model to feature number 353, P124.PWY..Bifidobacterium.shunt
## 2025-02-26 12:08:39.599798 INFO::Fitting model to feature number 354, PWY.622..starch.biosynthesis
## 2025-02-26 12:08:39.602119 INFO::Fitting model to feature number 355, PWY.7269..NAD.NADP.NADH.NADPH.mitochondrial.interconversion..yeast.
## 2025-02-26 12:08:39.604464 INFO::Fitting model to feature number 356, PWY.7316..dTDP.N.acetylviosamine.biosynthesis
## 2025-02-26 12:08:39.606808 INFO::Fitting model to feature number 357, PWY.7279..aerobic.respiration.II..cytochrome.c...yeast.
## 2025-02-26 12:08:39.609174 INFO::Fitting model to feature number 358, PWY.6837..fatty.acid.beta.oxidation.V..unsaturated..odd.number..di.isomerase.dependent.
## 2025-02-26 12:08:39.61149 INFO::Fitting model to feature number 359, X3.HYDROXYPHENYLACETATE.DEGRADATION.PWY..4.hydroxyphenylacetate.degradation
## 2025-02-26 12:08:39.613847 INFO::Fitting model to feature number 360, PWY.6953..dTDP.3.acetamido.3.6.dideoxy..alpha..D.galactose.biosynthesis
## 2025-02-26 12:08:39.616193 INFO::Fitting model to feature number 361, PWY.7616..methanol.oxidation.to.carbon.dioxide
## 2025-02-26 12:08:39.618601 INFO::Fitting model to feature number 362, PWY.5088..L.glutamate.degradation.VIII..to.propanoate.
## 2025-02-26 12:08:39.620935 INFO::Fitting model to feature number 363, PWY0.881..superpathway.of.fatty.acid.biosynthesis.I..E..coli.
## 2025-02-26 12:08:39.669578 INFO::Counting total values for each feature
## 2025-02-26 12:08:39.699257 INFO::Writing filtered data to file output_pwys/features/filtered_data.tsv
## 2025-02-26 12:08:39.726796 INFO::Writing filtered, normalized data to file output_pwys/features/filtered_data_norm.tsv
## 2025-02-26 12:08:39.753971 INFO::Writing filtered, normalized, transformed data to file output_pwys/features/filtered_data_norm_transformed.tsv
## 2025-02-26 12:08:39.790955 INFO::Writing residuals to file output_pwys/fits/residuals.rds
## 2025-02-26 12:08:39.812408 INFO::Writing fitted values to file output_pwys/fits/fitted.rds
## 2025-02-26 12:08:39.826912 INFO::Writing all results to file (ordered by increasing q-values): output_pwys/all_results.tsv
## 2025-02-26 12:08:39.832017 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
## 2025-02-26 12:08:39.832808 INFO::Writing heatmap of significant results to file: output_pwys/heatmap.pdf
## [1] "There are no associations to plot!"
## 2025-02-26 12:08:39.833788 INFO::Writing association plots (one for each significant association) to output folder: output_pwys
## 2025-02-26 12:08:39.83728 INFO::Plotting associations from most to least significant, grouped by metadata
## 2025-02-26 12:08:39.837882 INFO::Plotting data for metadata number 1, antibiotics_current_use
## 2025-02-26 12:08:39.839 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"