17  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).

17.1 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.

17.2 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) %>%
    .[, c("age", "disease", "antibiotics_current_use")]

#################
# Set reference #
#################

sample_metadata$disease <- as.factor(sample_metadata$disease)
sample_metadata$disease <- relevel(sample_metadata$disease, 'healthy')

###########################
# Create species features #
###########################

feature_species_t <- as.data.frame(assay(se_relative))
rownames(feature_species_t) <- sub('.*s__', '', rownames(feature_species_t))

##############################
# Subset to baseline samples #
##############################

feature_species <- as.data.frame(t(feature_species_t))
feature_species <- feature_species[rownames(sample_metadata),]
feature_species <- feature_species / 100
rm(feature_species_t); rm(se_relative)

In the next step, we will use MaAsLin2 to fit a multivariable regression model for testing the association between microbial species abundance versus IBD diagnosis. The analysis method we use here is “LM”, which is the default setting. We also adjust for age and antibiotic usage, following the original study.

library(Maaslin2)

fit_data = Maaslin2(
    input_data = feature_species,
    input_metadata = sample_metadata,
    normalization = "NONE",
    output = "output_species",
    fixed_effects = c("disease", "age", "antibiotics_current_use"))
##  [1] "Creating output folder"
##  [1] "Creating output feature tables folder"
##  [1] "Creating output fits folder"
##  [1] "Creating output figures folder"
##  2024-09-03 05:22:57.244783 INFO::Writing function arguments to log file
##  2024-09-03 05:22:57.259399 INFO::Verifying options selected are valid
##  2024-09-03 05:22:57.286589 INFO::Determining format of input files
##  2024-09-03 05:22:57.287105 INFO::Input format is data samples as rows and metadata samples as rows
##  2024-09-03 05:22:57.292904 INFO::Formula for fixed effects: expr ~  disease + age + antibiotics_current_use
##  2024-09-03 05:22:57.293573 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
##  2024-09-03 05:22:57.294164 INFO::Filter data based on min abundance and min prevalence
##  2024-09-03 05:22:57.294595 INFO::Total samples in data: 136
##  2024-09-03 05:22:57.295017 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
##  2024-09-03 05:22:57.300801 INFO::Total filtered features: 452
##  2024-09-03 05:22:57.301468 INFO::Filtered feature names from abundance and prevalence filtering: Abiotrophia.sp..HMSC24B09, Acidaminococcus.sp..CAG.542, Acinetobacter.lwoffii, Acinetobacter.ursingii, Actinobaculum.sp..oral.taxon.183, Actinomyces.graevenitzii, Actinomyces.sp..HMSC035G02, Actinomyces.sp..HPA0247, Actinomyces.sp..ICM47, Actinomyces.sp..oral.taxon.180, Actinomyces.sp..oral.taxon.181, Actinomyces.urogenitalis, Actinotignum.timonense, Adlercreutzia.caecimuris, Adlercreutzia.equolifaciens, Aeriscardovia.aeriphila, Aerococcus.urinaeequi, Aeromonas.dhakensis, Aeromonas.hydrophila, Aggregatibacter.aphrophilus, Aggregatibacter.segnis, Aggregatibacter.sp..oral.taxon.458, Alistipes.inops, Alistipes.onderdonkii, Alistipes.timonensis, Allisonella.histaminiformans, Alloprevotella.tannerae, Alloscardovia.omnicolens, Amedibacillus.dolichus, Anaerococcus.lactolyticus, Anaerococcus.vaginalis, Anaerocolumna.aminovalerica, Anaerofilum.sp..An201, Anaerofustis.stercorihominis, Anaeroglobus.geminatus, Anaeromassilibacillus.sp..An172, Anaerosporobacter.mobilis, Anaerostipes.caccae, Anaerostipes.sp..494a, Anaerostipes.sp..992a, Anaerotruncus.sp..CAG.528, Arthrospira.platensis, Atopobium.deltae, Atopobium.minutum, Bacteroidales.bacterium.KA00251, Bacteroides.clarus, Bacteroides.fluxus, Bacteroides.nordii, Bacteroides.oleiciplenus, Bacteroides.sp..43_108, Bacteroides.sp..CAG.144, Bacteroides.sp..CAG.530, Bacteroides.sp..CAG.598, Bacteroides.sp..CAG.633, Bacteroides.sp..CAG.661, Bacteroides.sp..D2, Bacteroides.sp..OM08.11, Bacteroides.stercorirosoris, Bacteroidetes.oral.taxon.274, Bavariicoccus.seileri, Bifidobacterium.angulatum, Bifidobacterium.animalis, Bifidobacterium.asteroides, Bifidobacterium.breve, Bifidobacterium.catenulatum, Bifidobacterium.dentium, Bifidobacterium.pseudolongum, Bifidobacterium.pullorum, Blastocystis.sp..subtype.1, Blautia.argi, Blautia.coccoides, Blautia.hansenii, Blautia.hydrogenotrophica, Blautia.producta, Brachyspira.pilosicoli, Butyricicoccus.pullicaecorum, Butyrivibrio.crossotus, Butyrivibrio.sp..CAG.318, Campylobacter.concisus, Campylobacter.gracilis, Campylobacter.hominis, Campylobacter.showae, Campylobacter.upsaliensis, Campylobacter.ureolyticus, Candidatus.Gastranaerophilales.bacterium, Candidatus.Methanomassiliicoccus.intestinalis, Candidatus.Stoquefichus.sp..KLE1796, Catenibacterium.mitsuokai, Cellulosilyticum.lentocellum, Chlamydia.ibidis, Christensenella.hongkongensis, Christensenella.minuta, Citrobacter.amalonaticus, Citrobacter.braakii, Citrobacter.europaeus, Citrobacter.farmeri, Citrobacter.freundii, Citrobacter.pasteurii, Citrobacter.portucalensis, Citrobacter.werkmanii, Citrobacter.youngae, Cloacibacillus.porcorum, Clostridiales.bacterium.1_7_47FAA, Clostridiales.bacterium.CHKCI006, Clostridioides.difficile, Clostridium.baratii, Clostridium.botulinum, Clostridium.butyricum, Clostridium.cadaveris, Clostridium.celatum, Clostridium.disporicum, Clostridium.neonatale, Clostridium.paraputrificum, Clostridium.perfringens, Clostridium.sp..7_2_43FAA, Clostridium.sp..CAG.167, Clostridium.sp..CAG.242, Clostridium.sp..CAG.253, Clostridium.sp..CAG.411, Clostridium.sp..CAG.413, Clostridium.sp..CAG.590, Clostridium.sp..CAG.678, Clostridium.sp..CAG.964, Clostridium.sp..D5, Clostridium.sp..MSTE9, Clostridium.sp..chh4.2, Clostridium.sporogenes, Comamonas.kerstersii, Coprobacillus.cateniformis, Coprobacter.secundus, Coprobacter.sp., Corynebacterium.accolens, Corynebacterium.amycolatum, Corynebacterium.kroppenstedtii, Corynebacterium.matruchotii, Corynebacterium.oculi, Cronobacter.malonaticus, Cronobacter.sakazakii, Cutibacterium.acnes, Cutibacterium.granulosum, Dellaglioa.algida, Desulfovibrio.fairfieldensis, Desulfovibrio.piger, Desulfovibrionaceae.bacterium, Dialister.micraerophilus, Dialister.pneumosintes, Dialister.sp..CAG.357, Dorea.sp..CAG.317, Dysgonomonas.gadei, Dysgonomonas.mossii, Dysgonomonas.sp..37.18, Eikenella.corrodens, Enhydrobacter.aerosaccus, Enorma.massiliensis, Enterobacter.mori, Enterocloster.asparagiformis, Enterococcus.asini, Enterococcus.avium, Enterococcus.casseliflavus, Enterococcus.dispar, Enterococcus.durans, Enterococcus.faecalis, Enterococcus.faecium, Enterococcus.gallinarum, Enterococcus.hirae, Enterococcus.mundtii, Enterococcus.pseudoavium, Enterococcus.raffinosus, Enterococcus.sp..3H8_DIV0648, Enterococcus.thailandicus, Erysipelothrix.larvae, Escherichia.albertii, Escherichia.marmotae, Eubacteriaceae.bacterium.CHKCI005, Eubacterium.coprostanoligenes, Eubacterium.dolichum.CAG.375, Eubacterium.limosum, Eubacterium.sp..An11, Eubacterium.sp..CAG.180, Eubacterium.sp..CAG.274, Eubacterium.sp..OM08.24, Ezakiella.coagulans, Faecalicatena.orotica, Faecalicoccus.pleomorphus, Faecalitalea.cylindroides, Fannyhessea.vaginae, Finegoldia.magna, Firmicutes.bacterium.CAG.110, Firmicutes.bacterium.CAG.145, Firmicutes.bacterium.CAG.170, Firmicutes.bacterium.CAG.238, Firmicutes.bacterium.CAG.424, Firmicutes.bacterium.CAG.534, Firmicutes.bacterium.CAG.646, Firmicutes.bacterium.CAG.95, Flavonifractor.sp..An10, Flavonifractor.sp..An100, Flavonifractor.sp..An306, Fretibacterium.fastidiosum, Frigoribacterium.sp..Leaf8, Fructilactobacillus.sanfranciscensis, Fusobacterium.equinum, Fusobacterium.gonidiaformans, Fusobacterium.mortiferum, Fusobacterium.naviforme, Fusobacterium.nucleatum, Fusobacterium.periodonticum, Fusobacterium.sp..CAG.439, Fusobacterium.sp..oral.taxon.370, Fusobacterium.ulcerans, Gemella.asaccharolytica, Gemella.haemolysans, Gemella.morbillorum, Gemella.sanguinis, Gemmiger.sp..An50, Gemmiger.sp..An87, Gleimia.europaea, Gordonibacter.pamelaeae, Granulicatella.adiacens, Haemophilus.haemolyticus, Haemophilus.influenzae, Haemophilus.parahaemolyticus, Haemophilus.paraphrohaemolyticus, Haemophilus.pittmaniae, Haemophilus.sputorum, Hafnia.alvei, Hafnia.paralvei, Harryflintia.acetispora, Holdemanella.biformis, Klebsiella.aerogenes, Klebsiella.michiganensis, Klebsiella.oxytoca, Klebsiella.quasipneumoniae, Klebsiella.variicola.CAG.634, Kluyvera.ascorbata, Kluyvera.cryocrescens, Kluyvera.georgiana, Kocuria.palustris, Kosakonia.sacchari, Lachnoclostridium.sp..An118, Lachnoclostridium.sp..An131, Lachnoclostridium.sp..An138, Lachnoclostridium.sp..An14, Lachnoclostridium.sp..An181, Lachnoclostridium.sp..An298, Lachnospiraceae.bacterium.2_1_46FAA, Lachnospiraceae.bacterium.oral.taxon.096, Lacrimispora.celerecrescens, Lacticaseibacillus.rhamnosus, Lactiplantibacillus.plantarum, Lactobacillus.acidophilus, Lactobacillus.amylovorus, Lactobacillus.crispatus, Lactobacillus.delbrueckii, Lactobacillus.gasseri, Lactobacillus.jensenii, Lactobacillus.johnsonii, Lactobacillus.paragasseri, Lactococcus.lactis, Lactococcus.petauri, Lactococcus.piscium, Lactonifactor.longoviformis, Lancefieldella.parvula, Lancefieldella.rimae, Latilactobacillus.sakei, Lawsonella.clevelandensis, Leclercia.adecarboxylata, Leuconostoc.garlicum, Leuconostoc.lactis, Ligilactobacillus.animalis, Ligilactobacillus.ruminis, Ligilactobacillus.salivarius, Limosilactobacillus.fermentum, Limosilactobacillus.mucosae, Limosilactobacillus.oris, Limosilactobacillus.reuteri, Limosilactobacillus.vaginalis, Massilimicrobiota.timonensis, Megamonas.funiformis, Megamonas.funiformis.CAG.377, Megamonas.hypermegale, Megasphaera.elsdenii, Megasphaera.micronuciformis, Megasphaera.sp..DISK.18, Megasphaera.sp..MJR8396C, Methanobrevibacter.smithii, Micrococcus.aloeverae, Micrococcus.luteus, Micrococcus.lylae, Microvirgula.aerodenitrificans, Mitsuokella.jalaludinii, Mitsuokella.multacida, Moraxella.osloensis, Morganella.morganii, Murimonas.intestini, Neisseria.cinerea, Neisseria.flavescens, Neisseria.sp..oral.taxon.014, Neisseria.subflava, Obesumbacterium.proteus, Odoribacter.laneus, Oscillibacter.sp..PC13, Oxalobacter.formigenes, Paenibacillus.macerans, Parabacteroides.goldsteinii, Parabacteroides.johnsonii, Parabacteroides.sp..CAG.409, Pararheinheimera.mesophila, Parvimonas.micra, Parvimonas.sp..KA00067, Parvimonas.sp..oral.taxon.110, Parvimonas.sp..oral.taxon.393, Paucilactobacillus.vaccinostercus, Pediococcus.acidilactici, Pediococcus.pentosaceus, Peptococcus.niger, Peptoniphilus.coxii, Peptoniphilus.duerdenii, Peptoniphilus.harei, Peptoniphilus.lacrimalis, Peptoniphilus.sp..BV3C26, Peptoniphilus.sp..HMSC062D09, Peptostreptococcus.anaerobius, Peptostreptococcus.stomatis, Phascolarctobacterium.sp..CAG.266, Phascolarctobacterium.succinatutens, Phocaeicola.sartorii, Phytobacter.palmae, Porphyromonas.asaccharolytica, Porphyromonas.endodontalis, Porphyromonas.somerae, Porphyromonas.sp..HMSC065F10, Porphyromonas.uenonis, Prevotella.amnii, Prevotella.bergensis, Prevotella.bivia, Prevotella.buccae, Prevotella.buccalis, Prevotella.colorans, Prevotella.corporis, Prevotella.dentalis, Prevotella.denticola, Prevotella.disiens, Prevotella.histicola, Prevotella.intermedia, Prevotella.jejuni, Prevotella.melaninogenica, Prevotella.nigrescens, Prevotella.oralis, Prevotella.oris, Prevotella.oulorum, Prevotella.pallens, Prevotella.salivae, Prevotella.sp..885, Prevotella.sp..AM42.24, Prevotella.sp..CAG.1092, Prevotella.sp..CAG.1185, Prevotella.sp..CAG.279, Prevotella.sp..CAG.520, Prevotella.sp..CAG.891, Prevotella.sp..S7.1.8, Prevotella.stercorea, Prevotella.timonensis, Proteus.hauseri, Proteus.mirabilis, Proteus.penneri, Proteus.vulgaris, Providencia.alcalifaciens, Pseudoflavonifractor.capillosus, Pseudoflavonifractor.sp..An184, Pseudoflavonifractor.sp..An85, Pseudomonas.fragi, Pseudomonas.guguanensis, Raoultella.ornithinolytica, Raoultella.planticola, Rikenella.microfusus, Robinsoniella.sp..RHS, Romboutsia.ilealis, Roseburia.sp..CAG.182, Roseburia.sp..CAG.303, Roseburia.sp..CAG.309, Rothia.mucilaginosa, Ruminococcaceae.bacterium.D5, Ruminococcus.callidus, Ruminococcus.obeum.CAG.39, Ruminococcus.sp..CAG.330, Ruminococcus.sp..CAG.403, Ruminococcus.sp..CAG.488, Ruminococcus.sp..CAG.563, Saccharomyces.cerevisiae, Sanguibacteroides.justesenii, Sarcina.ventriculi, Scardovia.wiggsiae, Schaalia.odontolytica, Schaalia.turicensis, Serratia.liquefaciens, Serratia.marcescens, Sharpea.azabuensis, Shuttleworthia.satelles, Slackia.isoflavoniconvertens, Solobacterium.moorei, Staphylococcus.aureus, Staphylococcus.epidermidis, Staphylococcus.hominis, Streptococcus.agalactiae, Streptococcus.australis, Streptococcus.cristatus, Streptococcus.gallolyticus, Streptococcus.gordonii, Streptococcus.infantis, Streptococcus.lutetiensis, Streptococcus.macedonicus, Streptococcus.milleri, Streptococcus.mitis, Streptococcus.mutans, Streptococcus.oralis, Streptococcus.pasteurianus, Streptococcus.peroris, Streptococcus.salivarius.CAG.79, Streptococcus.sanguinis, Streptococcus.sp..A12, Streptococcus.sp..F0442, Streptococcus.sp..HPH0090, Streptococcus.sp..M334, Streptococcus.thermophilus, Streptococcus.vestibularis, Terrisporobacter.othiniensis, Thermoleophilum.album, Tractidigestivibacter.scatoligenes, Treponema.lecithinolyticum, Turicibacter.sanguinis, Tyzzerella.nexilis, Varibaculum.cambriense, Veillonella.rodentium, Veillonella.rogosae, Veillonella.seminalis, Veillonella.sp..CAG.933, Veillonella.tobetsuensis, Victivallis.vadensis, Weissella.cibaria, Weissella.confusa, Weissella.viridescens, Wohlfahrtiimonas.chitiniclastica, Yersinia.frederiksenii, X.Bacteroides..pectinophilus, X.Butyribacterium..methylotrophicum, X.Clostridium..hylemonae, X.Clostridium..methylpentosum, X.Clostridium..scindens, X.Clostridium..spiroforme, X.Eubacterium..brachy, X.Eubacterium..infirmum
##  2024-09-03 05:22:57.304176 INFO::Total filtered features with variance filtering: 0
##  2024-09-03 05:22:57.304696 INFO::Filtered feature names from variance filtering:
##  2024-09-03 05:22:57.305117 INFO::Running selected normalization method: NONE
##  2024-09-03 05:22:57.30558 INFO::Applying z-score to standardize continuous metadata
##  2024-09-03 05:22:57.309697 INFO::Running selected transform method: LOG
##  2024-09-03 05:22:57.312503 INFO::Running selected analysis method: LM
##  2024-09-03 05:22:57.315933 INFO::Fitting model to feature number 1, Acidaminococcus.intestini
##  2024-09-03 05:22:57.322078 INFO::Fitting model to feature number 2, Agathobaculum.butyriciproducens
##  2024-09-03 05:22:57.324341 INFO::Fitting model to feature number 3, Akkermansia.muciniphila
##  2024-09-03 05:22:57.326615 INFO::Fitting model to feature number 4, Alistipes.finegoldii
##  2024-09-03 05:22:57.328898 INFO::Fitting model to feature number 5, Alistipes.indistinctus
##  2024-09-03 05:22:57.331102 INFO::Fitting model to feature number 6, Alistipes.putredinis
##  2024-09-03 05:22:57.333367 INFO::Fitting model to feature number 7, Alistipes.shahii
##  2024-09-03 05:22:57.33558 INFO::Fitting model to feature number 8, Anaerobutyricum.hallii
##  2024-09-03 05:22:57.337874 INFO::Fitting model to feature number 9, Anaeromassilibacillus.sp..An250
##  2024-09-03 05:22:57.340132 INFO::Fitting model to feature number 10, Anaerostipes.hadrus
##  2024-09-03 05:22:57.342422 INFO::Fitting model to feature number 11, Anaerotignum.lactatifermentans
##  2024-09-03 05:22:57.344665 INFO::Fitting model to feature number 12, Anaerotruncus.colihominis
##  2024-09-03 05:22:57.346889 INFO::Fitting model to feature number 13, Bacteroides.caccae
##  2024-09-03 05:22:57.349159 INFO::Fitting model to feature number 14, Bacteroides.cellulosilyticus
##  2024-09-03 05:22:57.351382 INFO::Fitting model to feature number 15, Bacteroides.eggerthii
##  2024-09-03 05:22:57.35364 INFO::Fitting model to feature number 16, Bacteroides.faecis
##  2024-09-03 05:22:57.355896 INFO::Fitting model to feature number 17, Bacteroides.faecis.CAG.32
##  2024-09-03 05:22:57.358208 INFO::Fitting model to feature number 18, Bacteroides.finegoldii
##  2024-09-03 05:22:57.360471 INFO::Fitting model to feature number 19, Bacteroides.fragilis
##  2024-09-03 05:22:57.362699 INFO::Fitting model to feature number 20, Bacteroides.galacturonicus
##  2024-09-03 05:22:57.365069 INFO::Fitting model to feature number 21, Bacteroides.intestinalis
##  2024-09-03 05:22:57.367331 INFO::Fitting model to feature number 22, Bacteroides.ovatus
##  2024-09-03 05:22:57.369669 INFO::Fitting model to feature number 23, Bacteroides.salyersiae
##  2024-09-03 05:22:57.371935 INFO::Fitting model to feature number 24, Bacteroides.stercoris
##  2024-09-03 05:22:57.374233 INFO::Fitting model to feature number 25, Bacteroides.thetaiotaomicron
##  2024-09-03 05:22:57.429048 INFO::Fitting model to feature number 26, Bacteroides.uniformis
##  2024-09-03 05:22:57.431526 INFO::Fitting model to feature number 27, Bacteroides.xylanisolvens
##  2024-09-03 05:22:57.433857 INFO::Fitting model to feature number 28, Barnesiella.intestinihominis
##  2024-09-03 05:22:57.436116 INFO::Fitting model to feature number 29, Bifidobacterium.adolescentis
##  2024-09-03 05:22:57.438402 INFO::Fitting model to feature number 30, Bifidobacterium.bifidum
##  2024-09-03 05:22:57.440689 INFO::Fitting model to feature number 31, Bifidobacterium.longum
##  2024-09-03 05:22:57.442925 INFO::Fitting model to feature number 32, Bifidobacterium.pseudocatenulatum
##  2024-09-03 05:22:57.445186 INFO::Fitting model to feature number 33, Bilophila.wadsworthia
##  2024-09-03 05:22:57.447484 INFO::Fitting model to feature number 34, Blautia.obeum
##  2024-09-03 05:22:57.449756 INFO::Fitting model to feature number 35, Blautia.sp..CAG.257
##  2024-09-03 05:22:57.451976 INFO::Fitting model to feature number 36, Blautia.wexlerae
##  2024-09-03 05:22:57.454228 INFO::Fitting model to feature number 37, Butyricimonas.synergistica
##  2024-09-03 05:22:57.456483 INFO::Fitting model to feature number 38, Butyricimonas.virosa
##  2024-09-03 05:22:57.458762 INFO::Fitting model to feature number 39, Clostridium.bolteae.CAG.59
##  2024-09-03 05:22:57.461018 INFO::Fitting model to feature number 40, Clostridium.sp..CAG.299
##  2024-09-03 05:22:57.463241 INFO::Fitting model to feature number 41, Clostridium.sp..CAG.58
##  2024-09-03 05:22:57.465529 INFO::Fitting model to feature number 42, Collinsella.aerofaciens
##  2024-09-03 05:22:57.467756 INFO::Fitting model to feature number 43, Collinsella.intestinalis
##  2024-09-03 05:22:57.469996 INFO::Fitting model to feature number 44, Coprobacter.fastidiosus
##  2024-09-03 05:22:57.472209 INFO::Fitting model to feature number 45, Coprococcus.catus
##  2024-09-03 05:22:57.474462 INFO::Fitting model to feature number 46, Coprococcus.comes
##  2024-09-03 05:22:57.476804 INFO::Fitting model to feature number 47, Coprococcus.eutactus
##  2024-09-03 05:22:57.479019 INFO::Fitting model to feature number 48, Dialister.invisus
##  2024-09-03 05:22:57.481271 INFO::Fitting model to feature number 49, Dielma.fastidiosa
##  2024-09-03 05:22:57.483492 INFO::Fitting model to feature number 50, Dorea.formicigenerans
##  2024-09-03 05:22:57.48575 INFO::Fitting model to feature number 51, Dorea.longicatena
##  2024-09-03 05:22:57.487968 INFO::Fitting model to feature number 52, Eggerthella.lenta
##  2024-09-03 05:22:57.490222 INFO::Fitting model to feature number 53, Eisenbergiella.massiliensis
##  2024-09-03 05:22:57.492462 INFO::Fitting model to feature number 54, Eisenbergiella.tayi
##  2024-09-03 05:22:57.49468 INFO::Fitting model to feature number 55, Enterocloster.aldenensis
##  2024-09-03 05:22:57.496933 INFO::Fitting model to feature number 56, Enterocloster.bolteae
##  2024-09-03 05:22:57.499161 INFO::Fitting model to feature number 57, Enterocloster.citroniae
##  2024-09-03 05:22:57.501408 INFO::Fitting model to feature number 58, Enterocloster.clostridioformis
##  2024-09-03 05:22:57.503653 INFO::Fitting model to feature number 59, Enterocloster.lavalensis
##  2024-09-03 05:22:57.505917 INFO::Fitting model to feature number 60, Erysipelatoclostridium.ramosum
##  2024-09-03 05:22:57.508251 INFO::Fitting model to feature number 61, Escherichia.coli
##  2024-09-03 05:22:57.510521 INFO::Fitting model to feature number 62, Eubacterium.ramulus
##  2024-09-03 05:22:57.512799 INFO::Fitting model to feature number 63, Eubacterium.sp..CAG.251
##  2024-09-03 05:22:57.51504 INFO::Fitting model to feature number 64, Eubacterium.sp..CAG.38
##  2024-09-03 05:22:57.517319 INFO::Fitting model to feature number 65, Eubacterium.ventriosum
##  2024-09-03 05:22:57.519543 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
##  2024-09-03 05:22:57.52179 INFO::Fitting model to feature number 67, Firmicutes.bacterium.CAG.83
##  2024-09-03 05:22:57.524008 INFO::Fitting model to feature number 68, Firmicutes.bacterium.CAG.94
##  2024-09-03 05:22:57.526254 INFO::Fitting model to feature number 69, Flavonifractor.plautii
##  2024-09-03 05:22:57.528511 INFO::Fitting model to feature number 70, Fusicatenibacter.saccharivorans
##  2024-09-03 05:22:57.530736 INFO::Fitting model to feature number 71, Gemmiger.formicilis
##  2024-09-03 05:22:57.532993 INFO::Fitting model to feature number 72, Haemophilus.parainfluenzae
##  2024-09-03 05:22:57.535225 INFO::Fitting model to feature number 73, Haemophilus.sp..HMSC71H05
##  2024-09-03 05:22:57.537484 INFO::Fitting model to feature number 74, Holdemania.filiformis
##  2024-09-03 05:22:57.539829 INFO::Fitting model to feature number 75, Hungatella.hathewayi
##  2024-09-03 05:22:57.542082 INFO::Fitting model to feature number 76, Intestinibacter.bartlettii
##  2024-09-03 05:22:57.544307 INFO::Fitting model to feature number 77, Intestinimonas.butyriciproducens
##  2024-09-03 05:22:57.546571 INFO::Fitting model to feature number 78, Klebsiella.pneumoniae
##  2024-09-03 05:22:57.548819 INFO::Fitting model to feature number 79, Klebsiella.variicola
##  2024-09-03 05:22:57.551037 INFO::Fitting model to feature number 80, Lachnospira.eligens
##  2024-09-03 05:22:57.553313 INFO::Fitting model to feature number 81, Lachnospira.pectinoschiza
##  2024-09-03 05:22:57.555541 INFO::Fitting model to feature number 82, Lacrimispora.saccharolytica
##  2024-09-03 05:22:57.557786 INFO::Fitting model to feature number 83, Lactobacillus.rogosae
##  2024-09-03 05:22:57.560021 INFO::Fitting model to feature number 84, Lawsonibacter.asaccharolyticus
##  2024-09-03 05:22:57.5623 INFO::Fitting model to feature number 85, Monoglobus.pectinilyticus
##  2024-09-03 05:22:57.564621 INFO::Fitting model to feature number 86, Odoribacter.splanchnicus
##  2024-09-03 05:22:57.566862 INFO::Fitting model to feature number 87, Oscillibacter.sp..57_20
##  2024-09-03 05:22:57.569131 INFO::Fitting model to feature number 88, Oscillibacter.sp..CAG.241
##  2024-09-03 05:22:57.571344 INFO::Fitting model to feature number 89, Parabacteroides.distasonis
##  2024-09-03 05:22:57.573599 INFO::Fitting model to feature number 90, Parabacteroides.merdae
##  2024-09-03 05:22:57.575819 INFO::Fitting model to feature number 91, Paraprevotella.clara
##  2024-09-03 05:22:57.578115 INFO::Fitting model to feature number 92, Paraprevotella.xylaniphila
##  2024-09-03 05:22:57.58034 INFO::Fitting model to feature number 93, Parasutterella.excrementihominis
##  2024-09-03 05:22:57.582603 INFO::Fitting model to feature number 94, Phascolarctobacterium.faecium
##  2024-09-03 05:22:57.58488 INFO::Fitting model to feature number 95, Phocaeicola.coprocola
##  2024-09-03 05:22:57.587131 INFO::Fitting model to feature number 96, Phocaeicola.dorei
##  2024-09-03 05:22:57.589407 INFO::Fitting model to feature number 97, Phocaeicola.massiliensis
##  2024-09-03 05:22:57.591631 INFO::Fitting model to feature number 98, Phocaeicola.plebeius
##  2024-09-03 05:22:57.5939 INFO::Fitting model to feature number 99, Phocaeicola.vulgatus
##  2024-09-03 05:22:57.596131 INFO::Fitting model to feature number 100, Prevotella.copri
##  2024-09-03 05:22:57.598399 INFO::Fitting model to feature number 101, Proteobacteria.bacterium.CAG.139
##  2024-09-03 05:22:57.600649 INFO::Fitting model to feature number 102, Roseburia.faecis
##  2024-09-03 05:22:57.602869 INFO::Fitting model to feature number 103, Roseburia.hominis
##  2024-09-03 05:22:57.605147 INFO::Fitting model to feature number 104, Roseburia.intestinalis
##  2024-09-03 05:22:57.607421 INFO::Fitting model to feature number 105, Roseburia.inulinivorans
##  2024-09-03 05:22:57.60968 INFO::Fitting model to feature number 106, Roseburia.sp..CAG.471
##  2024-09-03 05:22:57.611901 INFO::Fitting model to feature number 107, Ruminococcaceae.bacterium.D16
##  2024-09-03 05:22:57.614154 INFO::Fitting model to feature number 108, Ruminococcus.bicirculans
##  2024-09-03 05:22:57.616408 INFO::Fitting model to feature number 109, Ruminococcus.bromii
##  2024-09-03 05:22:57.618661 INFO::Fitting model to feature number 110, Ruthenibacterium.lactatiformans
##  2024-09-03 05:22:57.620946 INFO::Fitting model to feature number 111, Sellimonas.intestinalis
##  2024-09-03 05:22:57.623188 INFO::Fitting model to feature number 112, Streptococcus.parasanguinis
##  2024-09-03 05:22:57.625453 INFO::Fitting model to feature number 113, Streptococcus.salivarius
##  2024-09-03 05:22:57.62769 INFO::Fitting model to feature number 114, Turicimonas.muris
##  2024-09-03 05:22:57.62995 INFO::Fitting model to feature number 115, Veillonella.atypica
##  2024-09-03 05:22:57.632238 INFO::Fitting model to feature number 116, Veillonella.dispar
##  2024-09-03 05:22:57.634516 INFO::Fitting model to feature number 117, Veillonella.infantium
##  2024-09-03 05:22:57.636779 INFO::Fitting model to feature number 118, Veillonella.parvula
##  2024-09-03 05:22:57.639034 INFO::Fitting model to feature number 119, Veillonella.sp..T11011.6
##  2024-09-03 05:22:57.641305 INFO::Fitting model to feature number 120, X.Clostridium..innocuum
##  2024-09-03 05:22:57.643636 INFO::Fitting model to feature number 121, X.Clostridium..leptum
##  2024-09-03 05:22:57.645907 INFO::Fitting model to feature number 122, X.Clostridium..symbiosum
##  2024-09-03 05:22:57.648187 INFO::Fitting model to feature number 123, X.Eubacterium..rectale
##  2024-09-03 05:22:57.650551 INFO::Fitting model to feature number 124, X.Eubacterium..siraeum
##  2024-09-03 05:22:57.652817 INFO::Fitting model to feature number 125, X.Ruminococcus..gnavus
##  2024-09-03 05:22:57.655057 INFO::Fitting model to feature number 126, X.Ruminococcus..lactaris
##  2024-09-03 05:22:57.657416 INFO::Fitting model to feature number 127, X.Ruminococcus..torques
##  2024-09-03 05:22:57.708563 INFO::Counting total values for each feature
##  2024-09-03 05:22:57.720189 INFO::Writing filtered data to file output_species/features/filtered_data.tsv
##  2024-09-03 05:22:57.732512 INFO::Writing filtered, normalized data to file output_species/features/filtered_data_norm.tsv
##  2024-09-03 05:22:57.744345 INFO::Writing filtered, normalized, transformed data to file output_species/features/filtered_data_norm_transformed.tsv
##  2024-09-03 05:22:57.761634 INFO::Writing residuals to file output_species/fits/residuals.rds
##  2024-09-03 05:22:57.768045 INFO::Writing fitted values to file output_species/fits/fitted.rds
##  2024-09-03 05:22:57.77466 INFO::Writing all results to file (ordered by increasing q-values): output_species/all_results.tsv
##  2024-09-03 05:22:57.779219 INFO::Writing the significant results (those which are less than or equal to the threshold of 0.250000 ) to file (ordered by increasing q-values): output_species/significant_results.tsv
##  2024-09-03 05:22:57.780358 INFO::Writing heatmap of significant results to file: output_species/heatmap.pdf
##  2024-09-03 05:22:57.888698 INFO::Writing association plots (one for each significant association) to output folder: output_species
##  2024-09-03 05:22:57.89121 INFO::Plotting associations from most to least significant, grouped by metadata
##  2024-09-03 05:22:57.891739 INFO::Plotting data for metadata number 1, age
##  2024-09-03 05:22:57.892642 INFO::Creating scatter plot for continuous data, age vs Alistipes.indistinctus
##  2024-09-03 05:22:58.066133 INFO::Creating scatter plot for continuous data, age vs Lacrimispora.saccharolytica
##  2024-09-03 05:22:58.23895 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bicirculans
##  2024-09-03 05:22:58.401421 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
##  2024-09-03 05:22:58.527267 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
##  2024-09-03 05:22:58.647285 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
##  2024-09-03 05:22:58.773234 INFO::Creating scatter plot for continuous data, age vs Lachnospira.eligens
##  2024-09-03 05:22:58.895804 INFO::Creating scatter plot for continuous data, age vs X.Ruminococcus..gnavus
##  2024-09-03 05:22:59.0169 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.virosa
##  2024-09-03 05:22:59.154728 INFO::Creating scatter plot for continuous data, age vs Lawsonibacter.asaccharolyticus
##  2024-09-03 05:22:59.272308 INFO::Creating scatter plot for continuous data, age vs X.Eubacterium..siraeum
##  2024-09-03 05:22:59.39003 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.synergistica
##  2024-09-03 05:22:59.507959 INFO::Creating scatter plot for continuous data, age vs Monoglobus.pectinilyticus
##  2024-09-03 05:22:59.625904 INFO::Creating scatter plot for continuous data, age vs Veillonella.atypica
##  2024-09-03 05:23:01.184427 INFO::Plotting data for metadata number 2, disease
##  2024-09-03 05:23:01.185535 INFO::Creating boxplot for categorical data, disease vs Alistipes.putredinis
##  2024-09-03 05:23:01.334781 INFO::Creating boxplot for categorical data, disease vs Gemmiger.formicilis
##  2024-09-03 05:23:01.476547 INFO::Creating boxplot for categorical data, disease vs X.Ruminococcus..torques
##  2024-09-03 05:23:01.585653 INFO::Creating boxplot for categorical data, disease vs Ruminococcus.bicirculans
##  2024-09-03 05:23:01.694001 INFO::Creating boxplot for categorical data, disease vs Sellimonas.intestinalis
##  2024-09-03 05:23:01.801965 INFO::Creating boxplot for categorical data, disease vs X.Clostridium..leptum
##  2024-09-03 05:23:01.912427 INFO::Creating boxplot for categorical data, disease vs Alistipes.shahii
##  2024-09-03 05:23:02.965899 INFO::Plotting data for metadata number 3, antibiotics_current_use
##  2024-09-03 05:23:02.967036 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Coprobacter.fastidiosus
##  2024-09-03 05:23:03.092309 INFO::Creating boxplot for categorical data, antibiotics_current_use vs X.Eubacterium..rectale
##  2024-09-03 05:23:03.200659 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Agathobaculum.butyriciproducens

Unlike 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] FALSE

If 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_P

After 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  0.0791

In 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          12

The 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.

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 = '')

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.

17.3 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"

17.4 Performing the MSEA analysis with pathway relative abundance data

Next, we repeat the MSEA with the pathway relative abundance data from the iHMP project and follow the same steps as before.

##########################
# Load HMP2 pathway data #
##########################

se_pathway <- sampleMetadata |>
    filter(study_name == "HMP_2019_ibdmdb") |>
    returnSamples("pathway_abundance", rownames = "short")

##########################
# Create sample metadata #
##########################

sample_metadata <- colData(se_pathway) %>%
    as.data.frame() %>% filter(visit_number == 1) %>%
    dplyr::select("age", "disease", "antibiotics_current_use")

# Set reference
sample_metadata$disease <- as.factor(sample_metadata$disease)
sample_metadata$disease <- relevel(sample_metadata$disease, 'healthy')

###########################
# Create Pathway Features #
###########################

feature_pwys_t <- as.data.frame(assay(se_pathway))
feature_pwys_t <- rownames_to_column(feature_pwys_t, "ID")
feature_pwys_t <- feature_pwys_t %>%
    filter(!grepl("\\|", ID)) %>%
    filter(!ID %in% c('UNMAPPED', 'UNINTEGRATED')) %>%
    column_to_rownames('ID') %>%
    as.data.frame()

##############################
# Subset to baseline samples #
##############################

feature_pwys <- as.data.frame(t(feature_pwys_t))
feature_pwys <- feature_pwys[rownames(sample_metadata),]
feature_pwys <- feature_pwys / 100
rm(feature_pwys_t); rm(se_pathway)

As before, we first run a MaAsLin2 analysis using default settings and construct the modules using residuals from the MaAsLin2 models.

fit_data = Maaslin2(
    input_data = feature_pwys,
    input_metadata = sample_metadata,
    normalization = "NONE",
    output = "output_pwys",
    fixed_effects = c("disease", "age", "antibiotics_current_use"))
##  [1] "Creating output folder"
##  [1] "Creating output feature tables folder"
##  [1] "Creating output fits folder"
##  [1] "Creating output figures folder"
##  2024-09-03 05:23:27.704321 INFO::Writing function arguments to log file
##  2024-09-03 05:23:27.708101 INFO::Verifying options selected are valid
##  2024-09-03 05:23:27.708601 INFO::Determining format of input files
##  2024-09-03 05:23:27.709064 INFO::Input format is data samples as rows and metadata samples as rows
##  2024-09-03 05:23:27.714382 INFO::Formula for fixed effects: expr ~  disease + age + antibiotics_current_use
##  2024-09-03 05:23:27.714922 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
##  2024-09-03 05:23:27.715513 INFO::Filter data based on min abundance and min prevalence
##  2024-09-03 05:23:27.715943 INFO::Total samples in data: 136
##  2024-09-03 05:23:27.71635 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
##  2024-09-03 05:23:27.721796 INFO::Total filtered features: 113
##  2024-09-03 05:23:27.72235 INFO::Filtered feature names from abundance and prevalence filtering: PWY.5044..purine.nucleotides.degradation.I..plants., PROPFERM.PWY..L.alanine.fermentation.to.propanoate.and.acetate, PWY.6596..adenosine.nucleotides.degradation.I, PWY.5004..superpathway.of.L.citrulline.metabolism, UDPNACETYLGALSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.II, PWY66.367..ketogenesis, PWY.5392..reductive.TCA.cycle.II, PWY.101..photosynthesis.light.reactions, PWY.7031..protein.N.glycosylation..bacterial., PWY.5265..peptidoglycan.biosynthesis.II..staphylococci., PWY.7268..NAD.NADP.NADH.NADPH.cytosolic.interconversion..yeast., PWY.7165..L.ascorbate.biosynthesis.VI..engineered.pathway., CATECHOL.ORTHO.CLEAVAGE.PWY..catechol.degradation.to..beta..ketoadipate, PWY.5417..catechol.degradation.III..ortho.cleavage.pathway., PWY.5431..aromatic.compounds.degradation.via..beta..ketoadipate, PWY3DJ.35471..L.ascorbate.biosynthesis.IV, PWY.6185..4.methylcatechol.degradation..ortho.cleavage., PWY.7245..superpathway.NAD.NADP...NADH.NADPH.interconversion..yeast., PWY.5181..toluene.degradation.III..aerobic...via.p.cresol., PWY.6562..norspermidine.biosynthesis, PWY.7431..aromatic.biogenic.amine.degradation..bacteria., PWY.6307..L.tryptophan.degradation.X..mammalian..via.tryptamine., PWY.6313..serotonin.degradation, URSIN.PWY..ureide.biosynthesis, LIPASYN.PWY..phospholipases, DHGLUCONATE.PYR.CAT.PWY..glucose.degradation..oxidative., DENITRIFICATION.PWY..nitrate.reduction.I..denitrification., PWY.6662..superpathway.of.quinolone.and.alkylquinolone.biosynthesis, PWY.6660..2.heptyl.3.hydroxy.4.1H..quinolone.biosynthesis, PWY.6182..superpathway.of.salicylate.degradation, P165.PWY..superpathway.of.purines.degradation.in.plants, PWY66.388..fatty.acid..alpha..oxidation.III, TYRFUMCAT.PWY..L.tyrosine.degradation.I, PWY.5101..L.isoleucine.biosynthesis.II, PWY.6167..flavin.biosynthesis.II..archaea., PWY.6748..nitrate.reduction.VII..denitrification., PWY.7039..phosphatidate.metabolism..as.a.signaling.molecule, VALDEG.PWY..L.valine.degradation.I, PWY.5198..factor.420.biosynthesis, PWY.6215..4.chlorobenzoate.degradation, AEROBACTINSYN.PWY..aerobactin.biosynthesis, P562.PWY..myo.inositol.degradation.I, PWY.7409..phospholipid.remodeling..phosphatidylethanolamine..yeast., PWY.4722..creatinine.degradation.II, PWY.3801..sucrose.degradation.II..sucrose.synthase., PWY.7345..superpathway.of.anaerobic.sucrose.degradation, P125.PWY..superpathway.of..R.R..butanediol.biosynthesis, PWY.5994..palmitate.biosynthesis.I..animals.and.fungi., PWY.4321..L.glutamate.degradation.IV, PWY.7528..L.methionine.salvage.cycle.I..bacteria.and.plants., PWY.4361..S.methyl.5.thio..alpha..D.ribose.1.phosphate.degradation, PWY.1541..superpathway.of.taurine.degradation, PWY.5654..2.amino.3.carboxymuconate.semialdehyde.degradation.to.2.oxopentenoate, PWY.6210..2.aminophenol.degradation, PWY.6071..superpathway.of.phenylethylamine.degradation, PWY0.321..phenylacetate.degradation.I..aerobic., PWY.7200..superpathway.of.pyrimidine.deoxyribonucleoside.salvage, PWY.2221..Entner.Doudoroff.pathway.III..semi.phosphorylative., PWY.6992..1.5.anhydrofructose.degradation, PWY.7294..xylose.degradation.IV, PWY.6145..superpathway.of.sialic.acids.and.CMP.sialic.acids.biosynthesis, PWY.5180..toluene.degradation.I..aerobic...via.o.cresol., PWY.5182..toluene.degradation.II..aerobic...via.4.methylcatechol., PWY.5415..catechol.degradation.I..meta.cleavage.pathway., PWY.6785..hydrogen.production.VIII, PWY.5509..adenosylcobalamin.biosynthesis.from.cobyrinate.a.c.diamide.I, PWY.6641..superpathway.of.sulfolactate.degradation, PWY.7527..L.methionine.salvage.cycle.III, PWY.6396..superpathway.of.2.3.butanediol.biosynthesis, PWY.6467..Kdo.transfer.to.lipid.IVA.III..Chlamydia., X7ALPHADEHYDROX.PWY..cholate.degradation..bacteria..anaerobic., PWY.7374..1.4.dihydroxy.6.naphthoate.biosynthesis.I, PWY.6906..chitin.derivatives.degradation, PWY.5514..UDP.N.acetyl.D.galactosamine.biosynthesis.II, PWY.7317..superpathway.of.dTDP.glucose.derived.O.antigen.building.blocks.biosynthesis, PWY.7218..photosynthetic.3.hydroxybutanoate.biosynthesis..engineered., PHOTOALL.PWY..oxygenic.photosynthesis, PWY.6344..L.ornithine.degradation.II..Stickland.reaction., P621.PWY..nylon.6.oligomer.degradation, PWY.5028..L.histidine.degradation.II, CRNFORCAT.PWY..creatinine.degradation.I, PWY.6269..adenosylcobalamin.salvage.from.cobinamide.II, PWY.7389..superpathway.of.anaerobic.energy.metabolism..invertebrates., PWY.7384..anaerobic.energy.metabolism..invertebrates..mitochondrial., PWY.6165..chorismate.biosynthesis.II..archaea., PWY5F9.12..biphenyl.degradation, PWY.5647..2.nitrobenzoate.degradation.I, PWY.6138..CMP.N.acetylneuraminate.biosynthesis.I..eukaryotes., PWY.5910..superpathway.of.geranylgeranyldiphosphate.biosynthesis.I..via.mevalonate., PWY.922..mevalonate.pathway.I, PWY.6435..4.hydroxybenzoate.biosynthesis.V, PWY3O.1109..superpathway.of.4.hydroxybenzoate.biosynthesis..yeast., PWY.5754..4.hydroxybenzoate.biosynthesis.I..eukaryotes., PWY1G.0..mycothiol.biosynthesis, PWY.1501..mandelate.degradation.I, PWY.6107..chlorosalicylate.degradation, PWY.5534..propylene.degradation, PWY.7118..chitin.degradation.to.ethanol, PWY.7290..Escherichia.coli.serotype.O86.O.antigen.biosynthesis, PWY.181..photorespiration, PWY.1622..formaldehyde.assimilation.I..serine.pathway., CODH.PWY..reductive.acetyl.coenzyme.A.pathway, PWY.6349..CDP.archaeol.biosynthesis, PWY.6174..mevalonate.pathway.II..archaea., PWY.7286..7..3.amino.3.carboxypropyl..wyosine.biosynthesis, METHANOGENESIS.PWY..methanogenesis.from.H2.and.CO2, PWY.7391..isoprene.biosynthesis.II..engineered., PWY.5055..nicotinate.degradation.III, PWY.7399..methylphosphonate.degradation.II, P241.PWY..coenzyme.B.biosynthesis, PWY.5178..toluene.degradation.IV..aerobic...via.catechol., PWY.5420..catechol.degradation.II..meta.cleavage.pathway., PWY.5419..catechol.degradation.to.2.oxopent.4.enoate.II
##  2024-09-03 05:23:27.728575 INFO::Total filtered features with variance filtering: 0
##  2024-09-03 05:23:27.729075 INFO::Filtered feature names from variance filtering:
##  2024-09-03 05:23:27.729497 INFO::Running selected normalization method: NONE
##  2024-09-03 05:23:27.7299 INFO::Applying z-score to standardize continuous metadata
##  2024-09-03 05:23:27.733224 INFO::Running selected transform method: LOG
##  2024-09-03 05:23:27.741085 INFO::Running selected analysis method: LM
##  2024-09-03 05:23:27.741621 INFO::Fitting model to feature number 1, PWY.1042..glycolysis.IV..plant.cytosol.
##  2024-09-03 05:23:27.744193 INFO::Fitting model to feature number 2, DTDPRHAMSYN.PWY..dTDP.L.rhamnose.biosynthesis.I
##  2024-09-03 05:23:27.746523 INFO::Fitting model to feature number 3, PWY.5695..urate.biosynthesis.inosine.5..phosphate.degradation
##  2024-09-03 05:23:27.748821 INFO::Fitting model to feature number 4, PWY.6936..seleno.amino.acid.biosynthesis
##  2024-09-03 05:23:27.751088 INFO::Fitting model to feature number 5, ILEUSYN.PWY..L.isoleucine.biosynthesis.I..from.threonine.
##  2024-09-03 05:23:27.753398 INFO::Fitting model to feature number 6, PWY.7111..pyruvate.fermentation.to.isobutanol..engineered.
##  2024-09-03 05:23:27.755656 INFO::Fitting model to feature number 7, VALSYN.PWY..L.valine.biosynthesis
##  2024-09-03 05:23:27.758073 INFO::Fitting model to feature number 8, PWY.6609..adenine.and.adenosine.salvage.III
##  2024-09-03 05:23:27.760341 INFO::Fitting model to feature number 9, PWY.6737..starch.degradation.V
##  2024-09-03 05:23:27.762665 INFO::Fitting model to feature number 10, PWY.7219..adenosine.ribonucleotides.de.novo.biosynthesis
##  2024-09-03 05:23:27.765011 INFO::Fitting model to feature number 11, PWY0.1586..peptidoglycan.maturation..meso.diaminopimelate.containing.
##  2024-09-03 05:23:27.767295 INFO::Fitting model to feature number 12, ASPASN.PWY..superpathway.of.L.aspartate.and.L.asparagine.biosynthesis
##  2024-09-03 05:23:27.769634 INFO::Fitting model to feature number 13, PWY.5973..cis.vaccenate.biosynthesis
##  2024-09-03 05:23:27.771904 INFO::Fitting model to feature number 14, PWY.7221..guanosine.ribonucleotides.de.novo.biosynthesis
##  2024-09-03 05:23:27.774215 INFO::Fitting model to feature number 15, PWY.5686..UMP.biosynthesis
##  2024-09-03 05:23:27.776535 INFO::Fitting model to feature number 16, X1CMET2.PWY..N10.formyl.tetrahydrofolate.biosynthesis
##  2024-09-03 05:23:27.778803 INFO::Fitting model to feature number 17, PWY.6122..5.aminoimidazole.ribonucleotide.biosynthesis.II
##  2024-09-03 05:23:27.781099 INFO::Fitting model to feature number 18, PWY.6277..superpathway.of.5.aminoimidazole.ribonucleotide.biosynthesis
##  2024-09-03 05:23:27.783365 INFO::Fitting model to feature number 19, PWY.6385..peptidoglycan.biosynthesis.III..mycobacteria.
##  2024-09-03 05:23:27.785709 INFO::Fitting model to feature number 20, PWY.3841..folate.transformations.II
##  2024-09-03 05:23:27.787997 INFO::Fitting model to feature number 21, RIBOSYN2.PWY..flavin.biosynthesis.I..bacteria.and.plants.
##  2024-09-03 05:23:27.790361 INFO::Fitting model to feature number 22, PWY.6387..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.I..meso.diaminopimelate.containing.
##  2024-09-03 05:23:27.792676 INFO::Fitting model to feature number 23, THISYNARA.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.III..eukaryotes.
##  2024-09-03 05:23:27.79495 INFO::Fitting model to feature number 24, PWY.7663..gondoate.biosynthesis..anaerobic.
##  2024-09-03 05:23:27.797353 INFO::Fitting model to feature number 25, PWY.6386..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.II..lysine.containing.
##  2024-09-03 05:23:27.799629 INFO::Fitting model to feature number 26, PWY.6700..queuosine.biosynthesis
##  2024-09-03 05:23:27.801932 INFO::Fitting model to feature number 27, PEPTIDOGLYCANSYN.PWY..peptidoglycan.biosynthesis.I..meso.diaminopimelate.containing.
##  2024-09-03 05:23:27.804191 INFO::Fitting model to feature number 28, TRNA.CHARGING.PWY..tRNA.charging
##  2024-09-03 05:23:27.806465 INFO::Fitting model to feature number 29, PWY.6121..5.aminoimidazole.ribonucleotide.biosynthesis.I
##  2024-09-03 05:23:27.808774 INFO::Fitting model to feature number 30, HISTSYN.PWY..L.histidine.biosynthesis
##  2024-09-03 05:23:27.811037 INFO::Fitting model to feature number 31, PWY.7229..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.I
##  2024-09-03 05:23:27.81333 INFO::Fitting model to feature number 32, PWY.7199..pyrimidine.deoxyribonucleosides.salvage
##  2024-09-03 05:23:27.81563 INFO::Fitting model to feature number 33, PANTO.PWY..phosphopantothenate.biosynthesis.I
##  2024-09-03 05:23:27.817953 INFO::Fitting model to feature number 34, PWY.2942..L.lysine.biosynthesis.III
##  2024-09-03 05:23:27.820246 INFO::Fitting model to feature number 35, PWY.7237..myo...chiro..and.scillo.inositol.degradation
##  2024-09-03 05:23:27.822528 INFO::Fitting model to feature number 36, PWY.6168..flavin.biosynthesis.III..fungi.
##  2024-09-03 05:23:27.824822 INFO::Fitting model to feature number 37, COA.PWY.1..coenzyme.A.biosynthesis.II..mammalian.
##  2024-09-03 05:23:27.827075 INFO::Fitting model to feature number 38, PWY.5667..CDP.diacylglycerol.biosynthesis.I
##  2024-09-03 05:23:27.829356 INFO::Fitting model to feature number 39, PWY0.1319..CDP.diacylglycerol.biosynthesis.II
##  2024-09-03 05:23:27.831619 INFO::Fitting model to feature number 40, PWY.5097..L.lysine.biosynthesis.VI
##  2024-09-03 05:23:27.83392 INFO::Fitting model to feature number 41, ANAGLYCOLYSIS.PWY..glycolysis.III..from.glucose.
##  2024-09-03 05:23:27.836179 INFO::Fitting model to feature number 42, PWY.6123..inosine.5..phosphate.biosynthesis.I
##  2024-09-03 05:23:27.838467 INFO::Fitting model to feature number 43, ARGININE.SYN4.PWY..L.ornithine.de.novo..biosynthesis
##  2024-09-03 05:23:27.840763 INFO::Fitting model to feature number 44, PWY.6163..chorismate.biosynthesis.from.3.dehydroquinate
##  2024-09-03 05:23:27.843024 INFO::Fitting model to feature number 45, THRESYN.PWY..superpathway.of.L.threonine.biosynthesis
##  2024-09-03 05:23:27.845316 INFO::Fitting model to feature number 46, PYRIDNUCSYN.PWY..NAD.biosynthesis.I..from.aspartate.
##  2024-09-03 05:23:27.847571 INFO::Fitting model to feature number 47, PWY.6124..inosine.5..phosphate.biosynthesis.II
##  2024-09-03 05:23:27.849905 INFO::Fitting model to feature number 48, PWY.6147..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.I
##  2024-09-03 05:23:27.852173 INFO::Fitting model to feature number 49, PWY.7539..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.III..Chlamydia.
##  2024-09-03 05:23:27.854451 INFO::Fitting model to feature number 50, PWY.6151..S.adenosyl.L.methionine.cycle.I
##  2024-09-03 05:23:27.856751 INFO::Fitting model to feature number 51, SER.GLYSYN.PWY..superpathway.of.L.serine.and.glycine.biosynthesis.I
##  2024-09-03 05:23:27.859012 INFO::Fitting model to feature number 52, PWY.6126..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.II
##  2024-09-03 05:23:27.861296 INFO::Fitting model to feature number 53, PANTOSYN.PWY..pantothenate.and.coenzyme.A.biosynthesis.I
##  2024-09-03 05:23:27.863542 INFO::Fitting model to feature number 54, PWY.7228..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.I
##  2024-09-03 05:23:27.865825 INFO::Fitting model to feature number 55, COA.PWY..coenzyme.A.biosynthesis.I
##  2024-09-03 05:23:27.868078 INFO::Fitting model to feature number 56, PWY.4242..pantothenate.and.coenzyme.A.biosynthesis.III
##  2024-09-03 05:23:27.870357 INFO::Fitting model to feature number 57, PWY.6703..preQ0.biosynthesis
##  2024-09-03 05:23:27.87266 INFO::Fitting model to feature number 58, THISYN.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.I
##  2024-09-03 05:23:27.874906 INFO::Fitting model to feature number 59, HISDEG.PWY..L.histidine.degradation.I
##  2024-09-03 05:23:27.877187 INFO::Fitting model to feature number 60, PWY.5659..GDP.mannose.biosynthesis
##  2024-09-03 05:23:27.879443 INFO::Fitting model to feature number 61, PWY.5030..L.histidine.degradation.III
##  2024-09-03 05:23:27.881749 INFO::Fitting model to feature number 62, PWY.5484..glycolysis.II..from.fructose.6.phosphate.
##  2024-09-03 05:23:27.884027 INFO::Fitting model to feature number 63, PWY.3001..superpathway.of.L.isoleucine.biosynthesis.I
##  2024-09-03 05:23:27.886315 INFO::Fitting model to feature number 64, PWY.6125..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.II
##  2024-09-03 05:23:27.888613 INFO::Fitting model to feature number 65, TRPSYN.PWY..L.tryptophan.biosynthesis
##  2024-09-03 05:23:27.890867 INFO::Fitting model to feature number 66, GLYCOLYSIS..glycolysis.I..from.glucose.6.phosphate.
##  2024-09-03 05:23:27.893139 INFO::Fitting model to feature number 67, PWY.724..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.II
##  2024-09-03 05:23:27.895393 INFO::Fitting model to feature number 68, PWY.6897..thiamin.salvage.II
##  2024-09-03 05:23:27.897704 INFO::Fitting model to feature number 69, BRANCHED.CHAIN.AA.SYN.PWY..superpathway.of.branched.amino.acid.biosynthesis
##  2024-09-03 05:23:27.899957 INFO::Fitting model to feature number 70, CALVIN.PWY..Calvin.Benson.Bassham.cycle
##  2024-09-03 05:23:27.902226 INFO::Fitting model to feature number 71, PWY.841..superpathway.of.purine.nucleotides.de.novo.biosynthesis.I
##  2024-09-03 05:23:27.904508 INFO::Fitting model to feature number 72, COMPLETE.ARO.PWY..superpathway.of.aromatic.amino.acid.biosynthesis
##  2024-09-03 05:23:27.906814 INFO::Fitting model to feature number 73, PWY.7220..adenosine.deoxyribonucleotides.de.novo.biosynthesis.II
##  2024-09-03 05:23:27.909149 INFO::Fitting model to feature number 74, PWY.7222..guanosine.deoxyribonucleotides.de.novo.biosynthesis.II
##  2024-09-03 05:23:27.911408 INFO::Fitting model to feature number 75, DENOVOPURINE2.PWY..superpathway.of.purine.nucleotides.de.novo.biosynthesis.II
##  2024-09-03 05:23:27.913718 INFO::Fitting model to feature number 76, PWY.7357..thiamin.formation.from.pyrithiamine.and.oxythiamine..yeast.
##  2024-09-03 05:23:27.915982 INFO::Fitting model to feature number 77, PWY.5103..L.isoleucine.biosynthesis.III
##  2024-09-03 05:23:27.918299 INFO::Fitting model to feature number 78, ARO.PWY..chorismate.biosynthesis.I
##  2024-09-03 05:23:27.920609 INFO::Fitting model to feature number 79, PWY.7282..4.amino.2.methyl.5.phosphomethylpyrimidine.biosynthesis..yeast.
##  2024-09-03 05:23:27.922877 INFO::Fitting model to feature number 80, PWY0.845..superpathway.of.pyridoxal.5..phosphate.biosynthesis.and.salvage
##  2024-09-03 05:23:27.925158 INFO::Fitting model to feature number 81, ANAEROFRUCAT.PWY..homolactic.fermentation
##  2024-09-03 05:23:27.927423 INFO::Fitting model to feature number 82, PWY.6892..thiazole.biosynthesis.I..E..coli.
##  2024-09-03 05:23:27.92974 INFO::Fitting model to feature number 83, RHAMCAT.PWY..L.rhamnose.degradation.I
##  2024-09-03 05:23:27.931996 INFO::Fitting model to feature number 84, PYRIDOXSYN.PWY..pyridoxal.5..phosphate.biosynthesis.I
##  2024-09-03 05:23:27.934273 INFO::Fitting model to feature number 85, PWY.1269..CMP.3.deoxy.D.manno.octulosonate.biosynthesis.I
##  2024-09-03 05:23:27.936709 INFO::Fitting model to feature number 86, PHOSLIPSYN.PWY..superpathway.of.phospholipid.biosynthesis.I..bacteria.
##  2024-09-03 05:23:27.938982 INFO::Fitting model to feature number 87, CITRULBIO.PWY..L.citrulline.biosynthesis
##  2024-09-03 05:23:27.941377 INFO::Fitting model to feature number 88, NAGLIPASYN.PWY..lipid.IVA.biosynthesis
##  2024-09-03 05:23:27.943627 INFO::Fitting model to feature number 89, BIOTIN.BIOSYNTHESIS.PWY..biotin.biosynthesis.I
##  2024-09-03 05:23:27.945914 INFO::Fitting model to feature number 90, PWY.5154..L.arginine.biosynthesis.III..via.N.acetyl.L.citrulline.
##  2024-09-03 05:23:27.948218 INFO::Fitting model to feature number 91, FASYN.ELONG.PWY..fatty.acid.elongation....saturated
##  2024-09-03 05:23:27.950652 INFO::Fitting model to feature number 92, PENTOSE.P.PWY..pentose.phosphate.pathway
##  2024-09-03 05:23:27.95298 INFO::Fitting model to feature number 93, PWYG.321..mycolate.biosynthesis
##  2024-09-03 05:23:27.955264 INFO::Fitting model to feature number 94, PWY.6519..8.amino.7.oxononanoate.biosynthesis.I
##  2024-09-03 05:23:27.957644 INFO::Fitting model to feature number 95, PWY.7664..oleate.biosynthesis.IV..anaerobic.
##  2024-09-03 05:23:27.959929 INFO::Fitting model to feature number 96, PWY.4981..L.proline.biosynthesis.II..from.arginine.
##  2024-09-03 05:23:27.962219 INFO::Fitting model to feature number 97, GLYCOGENSYNTH.PWY..glycogen.biosynthesis.I..from.ADP.D.Glucose.
##  2024-09-03 05:23:27.964505 INFO::Fitting model to feature number 98, PWY.7388..octanoyl..acyl.carrier.protein..biosynthesis..mitochondria..yeast.
##  2024-09-03 05:23:27.966772 INFO::Fitting model to feature number 99, NONOXIPENT.PWY..pentose.phosphate.pathway..non.oxidative.branch.
##  2024-09-03 05:23:27.969084 INFO::Fitting model to feature number 100, COBALSYN.PWY..adenosylcobalamin.salvage.from.cobinamide.I
##  2024-09-03 05:23:27.971363 INFO::Fitting model to feature number 101, PWY.5989..stearate.biosynthesis.II..bacteria.and.plants.
##  2024-09-03 05:23:27.97366 INFO::Fitting model to feature number 102, PWY.6282..palmitoleate.biosynthesis.I..from..5Z..dodec.5.enoate.
##  2024-09-03 05:23:27.975926 INFO::Fitting model to feature number 103, PWY0.862...5Z..dodec.5.enoate.biosynthesis
##  2024-09-03 05:23:27.978243 INFO::Fitting model to feature number 104, PWY.7400..L.arginine.biosynthesis.IV..archaebacteria.
##  2024-09-03 05:23:27.980548 INFO::Fitting model to feature number 105, ARGSYN.PWY..L.arginine.biosynthesis.I..via.L.ornithine.
##  2024-09-03 05:23:27.982806 INFO::Fitting model to feature number 106, PWY.4984..urea.cycle
##  2024-09-03 05:23:27.985094 INFO::Fitting model to feature number 107, FASYN.INITIAL.PWY..superpathway.of.fatty.acid.biosynthesis.initiation..E..coli.
##  2024-09-03 05:23:27.98735 INFO::Fitting model to feature number 108, PWY4FS.7..phosphatidylglycerol.biosynthesis.I..plastidic.
##  2024-09-03 05:23:27.989638 INFO::Fitting model to feature number 109, PWY4FS.8..phosphatidylglycerol.biosynthesis.II..non.plastidic.
##  2024-09-03 05:23:27.991889 INFO::Fitting model to feature number 110, ARGSYNBSUB.PWY..L.arginine.biosynthesis.II..acetyl.cycle.
##  2024-09-03 05:23:27.994182 INFO::Fitting model to feature number 111, PWY.6317..galactose.degradation.I..Leloir.pathway.
##  2024-09-03 05:23:27.996461 INFO::Fitting model to feature number 112, PWY66.422..D.galactose.degradation.V..Leloir.pathway.
##  2024-09-03 05:23:27.998714 INFO::Fitting model to feature number 113, GLUTORN.PWY..L.ornithine.biosynthesis
##  2024-09-03 05:23:28.001134 INFO::Fitting model to feature number 114, PWY.2941..L.lysine.biosynthesis.II
##  2024-09-03 05:23:28.003401 INFO::Fitting model to feature number 115, PWY0.1296..purine.ribonucleosides.degradation
##  2024-09-03 05:23:28.005682 INFO::Fitting model to feature number 116, NAD.BIOSYNTHESIS.II..NAD.salvage.pathway.II
##  2024-09-03 05:23:28.007957 INFO::Fitting model to feature number 117, HSERMETANA.PWY..L.methionine.biosynthesis.III
##  2024-09-03 05:23:28.010293 INFO::Fitting model to feature number 118, PWY0.162..superpathway.of.pyrimidine.ribonucleotides.de.novo.biosynthesis
##  2024-09-03 05:23:28.012575 INFO::Fitting model to feature number 119, PWY.7208..superpathway.of.pyrimidine.nucleobases.salvage
##  2024-09-03 05:23:28.014825 INFO::Fitting model to feature number 120, TEICHOICACID.PWY..teichoic.acid..poly.glycerol..biosynthesis
##  2024-09-03 05:23:28.017104 INFO::Fitting model to feature number 121, PWY.6305..putrescine.biosynthesis.IV
##  2024-09-03 05:23:28.019342 INFO::Fitting model to feature number 122, GLUCONEO.PWY..gluconeogenesis.I
##  2024-09-03 05:23:28.021639 INFO::Fitting model to feature number 123, PWY.621..sucrose.degradation.III..sucrose.invertase.
##  2024-09-03 05:23:28.023892 INFO::Fitting model to feature number 124, PWY.6901..superpathway.of.glucose.and.xylose.degradation
##  2024-09-03 05:23:28.026175 INFO::Fitting model to feature number 125, POLYISOPRENSYN.PWY..polyisoprenoid.biosynthesis..E..coli.
##  2024-09-03 05:23:28.028438 INFO::Fitting model to feature number 126, PWY.7323..superpathway.of.GDP.mannose.derived.O.antigen.building.blocks.biosynthesis
##  2024-09-03 05:23:28.030835 INFO::Fitting model to feature number 127, PWY.5941..glycogen.degradation.II..eukaryotic.
##  2024-09-03 05:23:28.03314 INFO::Fitting model to feature number 128, PWY.6527..stachyose.degradation
##  2024-09-03 05:23:28.035359 INFO::Fitting model to feature number 129, PWY0.1261..anhydromuropeptides.recycling
##  2024-09-03 05:23:28.037646 INFO::Fitting model to feature number 130, PWY66.400..glycolysis.VI..metazoan.
##  2024-09-03 05:23:28.039978 INFO::Fitting model to feature number 131, PWY0.166..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis..E..coli.
##  2024-09-03 05:23:28.042305 INFO::Fitting model to feature number 132, GLYCOLYSIS.E.D..superpathway.of.glycolysis.and.Entner.Doudoroff
##  2024-09-03 05:23:28.044592 INFO::Fitting model to feature number 133, PWY.7187..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.II
##  2024-09-03 05:23:28.046867 INFO::Fitting model to feature number 134, PWY.7456..mannan.degradation
##  2024-09-03 05:23:28.04921 INFO::Fitting model to feature number 135, PWY.7184..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.I
##  2024-09-03 05:23:28.051519 INFO::Fitting model to feature number 136, OANTIGEN.PWY..O.antigen.building.blocks.biosynthesis..E..coli.
##  2024-09-03 05:23:28.053828 INFO::Fitting model to feature number 137, COLANSYN.PWY..colanic.acid.building.blocks.biosynthesis
##  2024-09-03 05:23:28.05612 INFO::Fitting model to feature number 138, PWY.6545..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.III
##  2024-09-03 05:23:28.058539 INFO::Fitting model to feature number 139, PWY.6895..superpathway.of.thiamin.diphosphate.biosynthesis.II
##  2024-09-03 05:23:28.060845 INFO::Fitting model to feature number 140, PWY.6608..guanosine.nucleotides.degradation.III
##  2024-09-03 05:23:28.063102 INFO::Fitting model to feature number 141, TCA..TCA.cycle.I..prokaryotic.
##  2024-09-03 05:23:28.065407 INFO::Fitting model to feature number 142, PWY.6859..all.trans.farnesol.biosynthesis
##  2024-09-03 05:23:28.067697 INFO::Fitting model to feature number 143, PWY.5100..pyruvate.fermentation.to.acetate.and.lactate.II
##  2024-09-03 05:23:28.069988 INFO::Fitting model to feature number 144, PWY.6572..chondroitin.sulfate.degradation.I..bacterial.
##  2024-09-03 05:23:28.072237 INFO::Fitting model to feature number 145, PWY.6471..peptidoglycan.biosynthesis.IV..Enterococcus.faecium.
##  2024-09-03 05:23:28.074522 INFO::Fitting model to feature number 146, PWY.6470..peptidoglycan.biosynthesis.V...beta..lactam.resistance.
##  2024-09-03 05:23:28.076791 INFO::Fitting model to feature number 147, PWY.5188..tetrapyrrole.biosynthesis.I..from.glutamate.
##  2024-09-03 05:23:28.079029 INFO::Fitting model to feature number 148, GLUCUROCAT.PWY..superpathway.of..beta..D.glucuronide.and.D.glucuronate.degradation
##  2024-09-03 05:23:28.081315 INFO::Fitting model to feature number 149, PWY.6749..CMP.legionaminate.biosynthesis.I
##  2024-09-03 05:23:28.083572 INFO::Fitting model to feature number 150, P164.PWY..purine.nucleobases.degradation.I..anaerobic.
##  2024-09-03 05:23:28.085862 INFO::Fitting model to feature number 151, PWY.7198..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.IV
##  2024-09-03 05:23:28.088145 INFO::Fitting model to feature number 152, PWY.7211..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis
##  2024-09-03 05:23:28.090466 INFO::Fitting model to feature number 153, PWY66.409..superpathway.of.purine.nucleotide.salvage
##  2024-09-03 05:23:28.092758 INFO::Fitting model to feature number 154, GALACT.GLUCUROCAT.PWY..superpathway.of.hexuronide.and.hexuronate.degradation
##  2024-09-03 05:23:28.095053 INFO::Fitting model to feature number 155, UDPNAGSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.I
##  2024-09-03 05:23:28.097365 INFO::Fitting model to feature number 156, PWY.6353..purine.nucleotides.degradation.II..aerobic.
##  2024-09-03 05:23:28.099654 INFO::Fitting model to feature number 157, PWY.7197..pyrimidine.deoxyribonucleotide.phosphorylation
##  2024-09-03 05:23:28.101967 INFO::Fitting model to feature number 158, PWY.7242..D.fructuronate.degradation
##  2024-09-03 05:23:28.104233 INFO::Fitting model to feature number 159, PWY.7196..superpathway.of.pyrimidine.ribonucleosides.salvage
##  2024-09-03 05:23:28.106521 INFO::Fitting model to feature number 160, PWY.5690..TCA.cycle.II..plants.and.fungi.
##  2024-09-03 05:23:28.108806 INFO::Fitting model to feature number 161, PWY.5177..glutaryl.CoA.degradation
##  2024-09-03 05:23:28.111078 INFO::Fitting model to feature number 162, PWY.6478..GDP.D.glycero..alpha..D.manno.heptose.biosynthesis
##  2024-09-03 05:23:28.11342 INFO::Fitting model to feature number 163, HEXITOLDEGSUPER.PWY..superpathway.of.hexitol.degradation..bacteria.
##  2024-09-03 05:23:28.115683 INFO::Fitting model to feature number 164, GALACTUROCAT.PWY..D.galacturonate.degradation.I
##  2024-09-03 05:23:28.117976 INFO::Fitting model to feature number 165, PWY.5104..L.isoleucine.biosynthesis.IV
##  2024-09-03 05:23:28.120299 INFO::Fitting model to feature number 166, PWY.5505..L.glutamate.and.L.glutamine.biosynthesis
##  2024-09-03 05:23:28.122605 INFO::Fitting model to feature number 167, P441.PWY..superpathway.of.N.acetylneuraminate.degradation
##  2024-09-03 05:23:28.124895 INFO::Fitting model to feature number 168, PWY.6606..guanosine.nucleotides.degradation.II
##  2024-09-03 05:23:28.127167 INFO::Fitting model to feature number 169, SALVADEHYPOX.PWY..adenosine.nucleotides.degradation.II
##  2024-09-03 05:23:28.129509 INFO::Fitting model to feature number 170, PWY.5005..biotin.biosynthesis.II
##  2024-09-03 05:23:28.13179 INFO::Fitting model to feature number 171, PWY.6549..L.glutamine.biosynthesis.III
##  2024-09-03 05:23:28.134069 INFO::Fitting model to feature number 172, GLCMANNANAUT.PWY..superpathway.of.N.acetylglucosamine..N.acetylmannosamine.and.N.acetylneuraminate.degradation
##  2024-09-03 05:23:28.136549 INFO::Fitting model to feature number 173, PWY0.1241..ADP.L.glycero..beta..D.manno.heptose.biosynthesis
##  2024-09-03 05:23:28.138827 INFO::Fitting model to feature number 174, PWY.6507..4.deoxy.L.threo.hex.4.enopyranuronate.degradation
##  2024-09-03 05:23:28.141264 INFO::Fitting model to feature number 175, GALACTARDEG.PWY..D.galactarate.degradation.I
##  2024-09-03 05:23:28.14358 INFO::Fitting model to feature number 176, GLUCARGALACTSUPER.PWY..superpathway.of.D.glucarate.and.D.galactarate.degradation
##  2024-09-03 05:23:28.145912 INFO::Fitting model to feature number 177, PWY.5676..acetyl.CoA.fermentation.to.butanoate.II
##  2024-09-03 05:23:28.148179 INFO::Fitting model to feature number 178, PWY.5347..superpathway.of.L.methionine.biosynthesis..transsulfuration.
##  2024-09-03 05:23:28.150576 INFO::Fitting model to feature number 179, PWY.5138..unsaturated..even.numbered.fatty.acid..beta..oxidation
##  2024-09-03 05:23:28.152936 INFO::Fitting model to feature number 180, MET.SAM.PWY..superpathway.of.S.adenosyl.L.methionine.biosynthesis
##  2024-09-03 05:23:28.155309 INFO::Fitting model to feature number 181, PWY66.399..gluconeogenesis.III
##  2024-09-03 05:23:28.157629 INFO::Fitting model to feature number 182, PWY0.1297..superpathway.of.purine.deoxyribonucleosides.degradation
##  2024-09-03 05:23:28.159916 INFO::Fitting model to feature number 183, METSYN.PWY..L.homoserine.and.L.methionine.biosynthesis
##  2024-09-03 05:23:28.162245 INFO::Fitting model to feature number 184, PWY.7332..superpathway.of.UDP.N.acetylglucosamine.derived.O.antigen.building.blocks.biosynthesis
##  2024-09-03 05:23:28.164563 INFO::Fitting model to feature number 185, PWY.6588..pyruvate.fermentation.to.acetone
##  2024-09-03 05:23:28.166852 INFO::Fitting model to feature number 186, PWY.6891..thiazole.biosynthesis.II..Bacillus.
##  2024-09-03 05:23:28.169202 INFO::Fitting model to feature number 187, GLUCARDEG.PWY..D.glucarate.degradation.I
##  2024-09-03 05:23:28.171485 INFO::Fitting model to feature number 188, METH.ACETATE.PWY..methanogenesis.from.acetate
##  2024-09-03 05:23:28.173797 INFO::Fitting model to feature number 189, PWY.7013..L.1.2.propanediol.degradation
##  2024-09-03 05:23:28.176086 INFO::Fitting model to feature number 190, PWY.6590..superpathway.of.Clostridium.acetobutylicum.acidogenic.fermentation
##  2024-09-03 05:23:28.17842 INFO::Fitting model to feature number 191, FAO.PWY..fatty.acid..beta..oxidation.I
##  2024-09-03 05:23:28.180737 INFO::Fitting model to feature number 192, PWY.6318..L.phenylalanine.degradation.IV..mammalian..via.side.chain.
##  2024-09-03 05:23:28.183074 INFO::Fitting model to feature number 193, CENTFERM.PWY..pyruvate.fermentation.to.butanoate
##  2024-09-03 05:23:28.185386 INFO::Fitting model to feature number 194, GOLPDLCAT.PWY..superpathway.of.glycerol.degradation.to.1.3.propanediol
##  2024-09-03 05:23:28.187685 INFO::Fitting model to feature number 195, PWY.5136..fatty.acid..beta..oxidation.II..peroxisome.
##  2024-09-03 05:23:28.189994 INFO::Fitting model to feature number 196, PWY66.391..fatty.acid..beta..oxidation.VI..peroxisome.
##  2024-09-03 05:23:28.192314 INFO::Fitting model to feature number 197, HOMOSER.METSYN.PWY..L.methionine.biosynthesis.I
##  2024-09-03 05:23:28.194646 INFO::Fitting model to feature number 198, METHGLYUT.PWY..superpathway.of.methylglyoxal.degradation
##  2024-09-03 05:23:28.19697 INFO::Fitting model to feature number 199, PWY.7288..fatty.acid..beta..oxidation..peroxisome..yeast.
##  2024-09-03 05:23:28.199288 INFO::Fitting model to feature number 200, PWY.7383..anaerobic.energy.metabolism..invertebrates..cytosol.
##  2024-09-03 05:23:28.201607 INFO::Fitting model to feature number 201, P162.PWY..L.glutamate.degradation.V..via.hydroxyglutarate.
##  2024-09-03 05:23:28.203886 INFO::Fitting model to feature number 202, PWY.4041...gamma..glutamyl.cycle
##  2024-09-03 05:23:28.206184 INFO::Fitting model to feature number 203, P108.PWY..pyruvate.fermentation.to.propanoate.I
##  2024-09-03 05:23:28.20855 INFO::Fitting model to feature number 204, PWY.5022..4.aminobutanoate.degradation.V
##  2024-09-03 05:23:28.210845 INFO::Fitting model to feature number 205, GLUDEG.I.PWY..GABA.shunt
##  2024-09-03 05:23:28.213162 INFO::Fitting model to feature number 206, PWY.5677..succinate.fermentation.to.butanoate
##  2024-09-03 05:23:28.215434 INFO::Fitting model to feature number 207, P163.PWY..L.lysine.fermentation.to.acetate.and.butanoate
##  2024-09-03 05:23:28.217774 INFO::Fitting model to feature number 208, PWY.3781..aerobic.respiration.I..cytochrome.c.
##  2024-09-03 05:23:28.220067 INFO::Fitting model to feature number 209, PWY.5083..NAD.NADH.phosphorylation.and.dephosphorylation
##  2024-09-03 05:23:28.222361 INFO::Fitting model to feature number 210, DAPLYSINESYN.PWY..L.lysine.biosynthesis.I
##  2024-09-03 05:23:28.224672 INFO::Fitting model to feature number 211, P461.PWY..hexitol.fermentation.to.lactate..formate..ethanol.and.acetate
##  2024-09-03 05:23:28.226956 INFO::Fitting model to feature number 212, PWY.1861..formaldehyde.assimilation.II..RuMP.Cycle.
##  2024-09-03 05:23:28.229288 INFO::Fitting model to feature number 213, PWY.7312..dTDP.D..beta..fucofuranose.biosynthesis
##  2024-09-03 05:23:28.231628 INFO::Fitting model to feature number 214, PWY.6630..superpathway.of.L.tyrosine.biosynthesis
##  2024-09-03 05:23:28.234513 INFO::Fitting model to feature number 215, PWY.6595..superpathway.of.guanosine.nucleotides.degradation..plants.
##  2024-09-03 05:23:28.237387 INFO::Fitting model to feature number 216, ARG.POLYAMINE.SYN..superpathway.of.arginine.and.polyamine.biosynthesis
##  2024-09-03 05:23:28.240246 INFO::Fitting model to feature number 217, PWY.7003..glycerol.degradation.to.butanol
##  2024-09-03 05:23:28.243103 INFO::Fitting model to feature number 218, P185.PWY..formaldehyde.assimilation.III..dihydroxyacetone.cycle.
##  2024-09-03 05:23:28.245982 INFO::Fitting model to feature number 219, POLYAMSYN.PWY..superpathway.of.polyamine.biosynthesis.I
##  2024-09-03 05:23:28.248854 INFO::Fitting model to feature number 220, PWY0.1298..superpathway.of.pyrimidine.deoxyribonucleosides.degradation
##  2024-09-03 05:23:28.251695 INFO::Fitting model to feature number 221, LACTOSECAT.PWY..lactose.and.galactose.degradation.I
##  2024-09-03 05:23:28.254966 INFO::Fitting model to feature number 222, PWY4LZ.257..superpathway.of.fermentation..Chlamydomonas.reinhardtii.
##  2024-09-03 05:23:28.25835 INFO::Fitting model to feature number 223, P161.PWY..acetylene.degradation
##  2024-09-03 05:23:28.261275 INFO::Fitting model to feature number 224, RUMP.PWY..formaldehyde.oxidation.I
##  2024-09-03 05:23:28.26412 INFO::Fitting model to feature number 225, PPGPPMET.PWY..ppGpp.biosynthesis
##  2024-09-03 05:23:28.266996 INFO::Fitting model to feature number 226, PWY.5304..superpathway.of.sulfur.oxidation..Acidianus.ambivalens.
##  2024-09-03 05:23:28.269881 INFO::Fitting model to feature number 227, PWY.6531..mannitol.cycle
##  2024-09-03 05:23:28.272758 INFO::Fitting model to feature number 228, FUC.RHAMCAT.PWY..superpathway.of.fucose.and.rhamnose.degradation
##  2024-09-03 05:23:28.275601 INFO::Fitting model to feature number 229, PWY.7315..dTDP.N.acetylthomosamine.biosynthesis
##  2024-09-03 05:23:28.278517 INFO::Fitting model to feature number 230, FUCCAT.PWY..fucose.degradation
##  2024-09-03 05:23:28.281404 INFO::Fitting model to feature number 231, PWY.7210..pyrimidine.deoxyribonucleotides.biosynthesis.from.CTP
##  2024-09-03 05:23:28.284286 INFO::Fitting model to feature number 232, FOLSYN.PWY..superpathway.of.tetrahydrofolate.biosynthesis.and.salvage
##  2024-09-03 05:23:28.287234 INFO::Fitting model to feature number 233, PWY.6612..superpathway.of.tetrahydrofolate.biosynthesis
##  2024-09-03 05:23:28.290133 INFO::Fitting model to feature number 234, PWY0.1061..superpathway.of.L.alanine.biosynthesis
##  2024-09-03 05:23:28.293 INFO::Fitting model to feature number 235, PWY.5189..tetrapyrrole.biosynthesis.II..from.glycine.
##  2024-09-03 05:23:28.295848 INFO::Fitting model to feature number 236, PWY.6876..isopropanol.biosynthesis
##  2024-09-03 05:23:28.298723 INFO::Fitting model to feature number 237, PWY0.781..aspartate.superpathway
##  2024-09-03 05:23:28.30158 INFO::Fitting model to feature number 238, P4.PWY..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.I
##  2024-09-03 05:23:28.304447 INFO::Fitting model to feature number 239, PWY0.1479..tRNA.processing
##  2024-09-03 05:23:28.307327 INFO::Fitting model to feature number 240, GLYCOLYSIS.TCA.GLYOX.BYPASS..superpathway.of.glycolysis..pyruvate.dehydrogenase..TCA..and.glyoxylate.bypass
##  2024-09-03 05:23:28.3102 INFO::Fitting model to feature number 241, PYRIDNUCSAL.PWY..NAD.salvage.pathway.I
##  2024-09-03 05:23:28.313078 INFO::Fitting model to feature number 242, GLYCOCAT.PWY..glycogen.degradation.I..bacterial.
##  2024-09-03 05:23:28.315924 INFO::Fitting model to feature number 243, PWY.5723..Rubisco.shunt
##  2024-09-03 05:23:28.31879 INFO::Fitting model to feature number 244, TCA.GLYOX.BYPASS..superpathway.of.glyoxylate.bypass.and.TCA
##  2024-09-03 05:23:28.321675 INFO::Fitting model to feature number 245, PWY.7385..1.3.propanediol.biosynthesis..engineered.
##  2024-09-03 05:23:28.324566 INFO::Fitting model to feature number 246, PWY.7254..TCA.cycle.VII..acetate.producers.
##  2024-09-03 05:23:28.328149 INFO::Fitting model to feature number 247, GLYOXYLATE.BYPASS..glyoxylate.cycle
##  2024-09-03 05:23:28.350116 INFO::Fitting model to feature number 248, PWY.6731..starch.degradation.III
##  2024-09-03 05:23:28.353076 INFO::Fitting model to feature number 249, PWY.7328..superpathway.of.UDP.glucose.derived.O.antigen.building.blocks.biosynthesis
##  2024-09-03 05:23:28.355949 INFO::Fitting model to feature number 250, POLYAMINSYN3.PWY..superpathway.of.polyamine.biosynthesis.II
##  2024-09-03 05:23:28.35885 INFO::Fitting model to feature number 251, PWY.2723..trehalose.degradation.V
##  2024-09-03 05:23:28.361743 INFO::Fitting model to feature number 252, GLUCOSE1PMETAB.PWY..glucose.and.glucose.1.phosphate.degradation
##  2024-09-03 05:23:28.364631 INFO::Fitting model to feature number 253, PWY.5384..sucrose.degradation.IV..sucrose.phosphorylase.
##  2024-09-03 05:23:28.367511 INFO::Fitting model to feature number 254, PWY.7046..4.coumarate.degradation..anaerobic.
##  2024-09-03 05:23:28.370413 INFO::Fitting model to feature number 255, PWY66.389..phytol.degradation
##  2024-09-03 05:23:28.373322 INFO::Fitting model to feature number 256, PWY.5971..palmitate.biosynthesis.II..bacteria.and.plants.
##  2024-09-03 05:23:28.376166 INFO::Fitting model to feature number 257, PWY.6629..superpathway.of.L.tryptophan.biosynthesis
##  2024-09-03 05:23:28.379058 INFO::Fitting model to feature number 258, PWY.5345..superpathway.of.L.methionine.biosynthesis..by.sulfhydrylation.
##  2024-09-03 05:23:28.381944 INFO::Fitting model to feature number 259, SULFATE.CYS.PWY..superpathway.of.sulfate.assimilation.and.cysteine.biosynthesis
##  2024-09-03 05:23:28.384811 INFO::Fitting model to feature number 260, PWY.5838..superpathway.of.menaquinol.8.biosynthesis.I
##  2024-09-03 05:23:28.387643 INFO::Fitting model to feature number 261, PRPP.PWY..superpathway.of.histidine..purine..and.pyrimidine.biosynthesis
##  2024-09-03 05:23:28.390518 INFO::Fitting model to feature number 262, PWY.5845..superpathway.of.menaquinol.9.biosynthesis
##  2024-09-03 05:23:28.393401 INFO::Fitting model to feature number 263, PWY.6113..superpathway.of.mycolate.biosynthesis
##  2024-09-03 05:23:28.396228 INFO::Fitting model to feature number 264, PWY.5897..superpathway.of.menaquinol.11.biosynthesis
##  2024-09-03 05:23:28.399101 INFO::Fitting model to feature number 265, PWY.5898..superpathway.of.menaquinol.12.biosynthesis
##  2024-09-03 05:23:28.402024 INFO::Fitting model to feature number 266, PWY.5899..superpathway.of.menaquinol.13.biosynthesis
##  2024-09-03 05:23:28.404905 INFO::Fitting model to feature number 267, PWY.7117..C4.photosynthetic.carbon.assimilation.cycle..PEPCK.type
##  2024-09-03 05:23:28.435717 INFO::Fitting model to feature number 268, PWY.6969..TCA.cycle.V..2.oxoglutarate.ferredoxin.oxidoreductase.
##  2024-09-03 05:23:28.438365 INFO::Fitting model to feature number 269, PWY.6628..superpathway.of.L.phenylalanine.biosynthesis
##  2024-09-03 05:23:28.440677 INFO::Fitting model to feature number 270, PWY.5861..superpathway.of.demethylmenaquinol.8.biosynthesis
##  2024-09-03 05:23:28.44293 INFO::Fitting model to feature number 271, PWY.5862..superpathway.of.demethylmenaquinol.9.biosynthesis
##  2024-09-03 05:23:28.445228 INFO::Fitting model to feature number 272, FERMENTATION.PWY..mixed.acid.fermentation
##  2024-09-03 05:23:28.4475 INFO::Fitting model to feature number 273, PWY.241..C4.photosynthetic.carbon.assimilation.cycle..NADP.ME.type
##  2024-09-03 05:23:28.449801 INFO::Fitting model to feature number 274, SO4ASSIM.PWY..sulfate.reduction.I..assimilatory.
##  2024-09-03 05:23:28.452135 INFO::Fitting model to feature number 275, KETOGLUCONMET.PWY..ketogluconate.metabolism
##  2024-09-03 05:23:28.454434 INFO::Fitting model to feature number 276, PWY.5840..superpathway.of.menaquinol.7.biosynthesis
##  2024-09-03 05:23:28.456722 INFO::Fitting model to feature number 277, PWY.5850..superpathway.of.menaquinol.6.biosynthesis.I
##  2024-09-03 05:23:28.459002 INFO::Fitting model to feature number 278, PWY.5896..superpathway.of.menaquinol.10.biosynthesis
##  2024-09-03 05:23:28.46131 INFO::Fitting model to feature number 279, P105.PWY..TCA.cycle.IV..2.oxoglutarate.decarboxylase.
##  2024-09-03 05:23:28.463651 INFO::Fitting model to feature number 280, PWY.5913..TCA.cycle.VI..obligate.autotrophs.
##  2024-09-03 05:23:28.465946 INFO::Fitting model to feature number 281, PWY.561..superpathway.of.glyoxylate.cycle.and.fatty.acid.degradation
##  2024-09-03 05:23:28.468218 INFO::Fitting model to feature number 282, PWY.4702..phytate.degradation.I
##  2024-09-03 05:23:28.470536 INFO::Fitting model to feature number 283, PWY.6803..phosphatidylcholine.acyl.editing
##  2024-09-03 05:23:28.472829 INFO::Fitting model to feature number 284, PWY.7115..C4.photosynthetic.carbon.assimilation.cycle..NAD.ME.type
##  2024-09-03 05:23:28.47509 INFO::Fitting model to feature number 285, PWY.5918..superpathay.of.heme.biosynthesis.from.glutamate
##  2024-09-03 05:23:28.477511 INFO::Fitting model to feature number 286, PWY.7234..inosine.5..phosphate.biosynthesis.III
##  2024-09-03 05:23:28.480882 INFO::Fitting model to feature number 287, PWY.6285..superpathway.of.fatty.acids.biosynthesis..E..coli.
##  2024-09-03 05:23:28.484254 INFO::Fitting model to feature number 288, PWY.5860..superpathway.of.demethylmenaquinol.6.biosynthesis.I
##  2024-09-03 05:23:28.487765 INFO::Fitting model to feature number 289, PWY.5863..superpathway.of.phylloquinol.biosynthesis
##  2024-09-03 05:23:28.491371 INFO::Fitting model to feature number 290, HEME.BIOSYNTHESIS.II..heme.biosynthesis.I..aerobic.
##  2024-09-03 05:23:28.494534 INFO::Fitting model to feature number 291, PWY.821..superpathway.of.sulfur.amino.acid.biosynthesis..Saccharomyces.cerevisiae.
##  2024-09-03 05:23:28.496884 INFO::Fitting model to feature number 292, PWY.5675..nitrate.reduction.V..assimilatory.
##  2024-09-03 05:23:28.499172 INFO::Fitting model to feature number 293, PWY.5791..1.4.dihydroxy.2.naphthoate.biosynthesis.II..plants.
##  2024-09-03 05:23:28.50166 INFO::Fitting model to feature number 294, PWY.5837..1.4.dihydroxy.2.naphthoate.biosynthesis.I
##  2024-09-03 05:23:28.503946 INFO::Fitting model to feature number 295, PWY.5920..superpathway.of.heme.biosynthesis.from.glycine
##  2024-09-03 05:23:28.506279 INFO::Fitting model to feature number 296, PWY.7204..pyridoxal.5..phosphate.salvage.II..plants.
##  2024-09-03 05:23:28.508613 INFO::Fitting model to feature number 297, ENTBACSYN.PWY..enterobactin.biosynthesis
##  2024-09-03 05:23:28.510894 INFO::Fitting model to feature number 298, PWY0.1415..superpathway.of.heme.biosynthesis.from.uroporphyrinogen.III
##  2024-09-03 05:23:28.513241 INFO::Fitting model to feature number 299, HEMESYN2.PWY..heme.biosynthesis.II..anaerobic.
##  2024-09-03 05:23:28.515524 INFO::Fitting model to feature number 300, PWY.6284..superpathway.of.unsaturated.fatty.acids.biosynthesis..E..coli.
##  2024-09-03 05:23:28.517864 INFO::Fitting model to feature number 301, P42.PWY..incomplete.reductive.TCA.cycle
##  2024-09-03 05:23:28.520138 INFO::Fitting model to feature number 302, PWY.5656..mannosylglycerate.biosynthesis.I
##  2024-09-03 05:23:28.522449 INFO::Fitting model to feature number 303, PWY.5173..superpathway.of.acetyl.CoA.biosynthesis
##  2024-09-03 05:23:28.524755 INFO::Fitting model to feature number 304, ECASYN.PWY..enterobacterial.common.antigen.biosynthesis
##  2024-09-03 05:23:28.527017 INFO::Fitting model to feature number 305, AST.PWY..L.arginine.degradation.II..AST.pathway.
##  2024-09-03 05:23:28.529332 INFO::Fitting model to feature number 306, PWY3O.355..stearate.biosynthesis.III..fungi.
##  2024-09-03 05:23:28.531603 INFO::Fitting model to feature number 307, PWY0.42..2.methylcitrate.cycle.I
##  2024-09-03 05:23:28.533908 INFO::Fitting model to feature number 308, PWY.7094..fatty.acid.salvage
##  2024-09-03 05:23:28.536177 INFO::Fitting model to feature number 309, PWY.5747..2.methylcitrate.cycle.II
##  2024-09-03 05:23:28.538553 INFO::Fitting model to feature number 310, PWY.5367..petroselinate.biosynthesis
##  2024-09-03 05:23:28.540864 INFO::Fitting model to feature number 311, PWY0.1533..methylphosphonate.degradation.I
##  2024-09-03 05:23:28.543151 INFO::Fitting model to feature number 312, PWY.6823..molybdenum.cofactor.biosynthesis
##  2024-09-03 05:23:28.54546 INFO::Fitting model to feature number 313, PWY.5705..allantoin.degradation.to.glyoxylate.III
##  2024-09-03 05:23:28.547732 INFO::Fitting model to feature number 314, LPSSYN.PWY..superpathway.of.lipopolysaccharide.biosynthesis
##  2024-09-03 05:23:28.550056 INFO::Fitting model to feature number 315, REDCITCYC..TCA.cycle.VIII..helicobacter.
##  2024-09-03 05:23:28.552327 INFO::Fitting model to feature number 316, GLYCOL.GLYOXDEG.PWY..superpathway.of.glycol.metabolism.and.degradation
##  2024-09-03 05:23:28.554628 INFO::Fitting model to feature number 317, P122.PWY..heterolactic.fermentation
##  2024-09-03 05:23:28.556921 INFO::Fitting model to feature number 318, KDO.NAGLIPASYN.PWY..superpathway.of..Kdo.2.lipid.A.biosynthesis
##  2024-09-03 05:23:28.559201 INFO::Fitting model to feature number 319, PWY.7446..sulfoglycolysis
##  2024-09-03 05:23:28.561512 INFO::Fitting model to feature number 320, PWY490.3..nitrate.reduction.VI..assimilatory.
##  2024-09-03 05:23:28.563782 INFO::Fitting model to feature number 321, PWY.7209..superpathway.of.pyrimidine.ribonucleosides.degradation
##  2024-09-03 05:23:28.566072 INFO::Fitting model to feature number 322, UBISYN.PWY..superpathway.of.ubiquinol.8.biosynthesis..prokaryotic.
##  2024-09-03 05:23:28.568345 INFO::Fitting model to feature number 323, PWY0.41..allantoin.degradation.IV..anaerobic.
##  2024-09-03 05:23:28.570653 INFO::Fitting model to feature number 324, PWY.5855..ubiquinol.7.biosynthesis..prokaryotic.
##  2024-09-03 05:23:28.572949 INFO::Fitting model to feature number 325, PWY.5856..ubiquinol.9.biosynthesis..prokaryotic.
##  2024-09-03 05:23:28.575214 INFO::Fitting model to feature number 326, PWY.5857..ubiquinol.10.biosynthesis..prokaryotic.
##  2024-09-03 05:23:28.577504 INFO::Fitting model to feature number 327, PWY.6708..ubiquinol.8.biosynthesis..prokaryotic.
##  2024-09-03 05:23:28.579777 INFO::Fitting model to feature number 328, PWY.5692..allantoin.degradation.to.glyoxylate.II
##  2024-09-03 05:23:28.58207 INFO::Fitting model to feature number 329, URDEGR.PWY..superpathway.of.allantoin.degradation.in.plants
##  2024-09-03 05:23:28.584313 INFO::Fitting model to feature number 330, PWY0.1338..polymyxin.resistance
##  2024-09-03 05:23:28.586625 INFO::Fitting model to feature number 331, P221.PWY..octane.oxidation
##  2024-09-03 05:23:28.588913 INFO::Fitting model to feature number 332, THREOCAT.PWY..superpathway.of.L.threonine.metabolism
##  2024-09-03 05:23:28.591176 INFO::Fitting model to feature number 333, NONMEVIPP.PWY..methylerythritol.phosphate.pathway.I
##  2024-09-03 05:23:28.593473 INFO::Fitting model to feature number 334, PWY.7560..methylerythritol.phosphate.pathway.II
##  2024-09-03 05:23:28.595736 INFO::Fitting model to feature number 335, PWY.5121..superpathway.of.geranylgeranyl.diphosphate.biosynthesis.II..via.MEP.
##  2024-09-03 05:23:28.598019 INFO::Fitting model to feature number 336, PWY.7392..taxadiene.biosynthesis..engineered.
##  2024-09-03 05:23:28.600282 INFO::Fitting model to feature number 337, PWY.6143..CMP.pseudaminate.biosynthesis
##  2024-09-03 05:23:28.602562 INFO::Fitting model to feature number 338, PWY.6270..isoprene.biosynthesis.I
##  2024-09-03 05:23:28.604901 INFO::Fitting model to feature number 339, ALLANTOINDEG.PWY..superpathway.of.allantoin.degradation.in.yeast
##  2024-09-03 05:23:28.607174 INFO::Fitting model to feature number 340, PWY.6263..superpathway.of.menaquinol.8.biosynthesis.II
##  2024-09-03 05:23:28.609452 INFO::Fitting model to feature number 341, PWY.7371..1.4.dihydroxy.6.naphthoate.biosynthesis.II
##  2024-09-03 05:23:28.611716 INFO::Fitting model to feature number 342, ARGORNPROST.PWY..arginine..ornithine.and.proline.interconversion
##  2024-09-03 05:23:28.613994 INFO::Fitting model to feature number 343, PWY66.398..TCA.cycle.III..animals.
##  2024-09-03 05:23:28.616251 INFO::Fitting model to feature number 344, PWY.5464..superpathway.of.cytosolic.glycolysis..plants...pyruvate.dehydrogenase.and.TCA.cycle
##  2024-09-03 05:23:28.618532 INFO::Fitting model to feature number 345, ORNDEG.PWY..superpathway.of.ornithine.degradation
##  2024-09-03 05:23:28.62088 INFO::Fitting model to feature number 346, P23.PWY..reductive.TCA.cycle.I
##  2024-09-03 05:23:28.623144 INFO::Fitting model to feature number 347, PWY0.1277..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation
##  2024-09-03 05:23:28.625451 INFO::Fitting model to feature number 348, ARGDEG.PWY..superpathway.of.L.arginine..putrescine..and.4.aminobutanoate.degradation
##  2024-09-03 05:23:28.627707 INFO::Fitting model to feature number 349, ORNARGDEG.PWY..superpathway.of.L.arginine.and.L.ornithine.degradation
##  2024-09-03 05:23:28.630014 INFO::Fitting model to feature number 350, HCAMHPDEG.PWY..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation.to.2.oxopent.4.enoate
##  2024-09-03 05:23:28.632261 INFO::Fitting model to feature number 351, PWY.6690..cinnamate.and.3.hydroxycinnamate.degradation.to.2.oxopent.4.enoate
##  2024-09-03 05:23:28.634544 INFO::Fitting model to feature number 352, PROTOCATECHUATE.ORTHO.CLEAVAGE.PWY..protocatechuate.degradation.II..ortho.cleavage.pathway.
##  2024-09-03 05:23:28.63684 INFO::Fitting model to feature number 353, P124.PWY..Bifidobacterium.shunt
##  2024-09-03 05:23:28.639096 INFO::Fitting model to feature number 354, PWY.622..starch.biosynthesis
##  2024-09-03 05:23:28.641391 INFO::Fitting model to feature number 355, PWY.7269..NAD.NADP.NADH.NADPH.mitochondrial.interconversion..yeast.
##  2024-09-03 05:23:28.643645 INFO::Fitting model to feature number 356, PWY.7316..dTDP.N.acetylviosamine.biosynthesis
##  2024-09-03 05:23:28.64594 INFO::Fitting model to feature number 357, PWY.7279..aerobic.respiration.II..cytochrome.c...yeast.
##  2024-09-03 05:23:28.6482 INFO::Fitting model to feature number 358, PWY.6837..fatty.acid.beta.oxidation.V..unsaturated..odd.number..di.isomerase.dependent.
##  2024-09-03 05:23:28.65049 INFO::Fitting model to feature number 359, X3.HYDROXYPHENYLACETATE.DEGRADATION.PWY..4.hydroxyphenylacetate.degradation
##  2024-09-03 05:23:28.65279 INFO::Fitting model to feature number 360, PWY.6953..dTDP.3.acetamido.3.6.dideoxy..alpha..D.galactose.biosynthesis
##  2024-09-03 05:23:28.655064 INFO::Fitting model to feature number 361, PWY.7616..methanol.oxidation.to.carbon.dioxide
##  2024-09-03 05:23:28.6574 INFO::Fitting model to feature number 362, PWY.5088..L.glutamate.degradation.VIII..to.propanoate.
##  2024-09-03 05:23:28.659672 INFO::Fitting model to feature number 363, PWY0.881..superpathway.of.fatty.acid.biosynthesis.I..E..coli.
##  2024-09-03 05:23:28.708015 INFO::Counting total values for each feature
##  2024-09-03 05:23:28.738889 INFO::Writing filtered data to file output_pwys/features/filtered_data.tsv
##  2024-09-03 05:23:28.776606 INFO::Writing filtered, normalized data to file output_pwys/features/filtered_data_norm.tsv
##  2024-09-03 05:23:28.814203 INFO::Writing filtered, normalized, transformed data to file output_pwys/features/filtered_data_norm_transformed.tsv
##  2024-09-03 05:23:28.862917 INFO::Writing residuals to file output_pwys/fits/residuals.rds
##  2024-09-03 05:23:28.88657 INFO::Writing fitted values to file output_pwys/fits/fitted.rds
##  2024-09-03 05:23:28.902753 INFO::Writing all results to file (ordered by increasing q-values): output_pwys/all_results.tsv
##  2024-09-03 05:23:28.908831 INFO::Writing the significant results (those which are less than or equal to the threshold of 0.250000 ) to file (ordered by increasing q-values): output_pwys/significant_results.tsv
##  2024-09-03 05:23:28.909596 INFO::Writing heatmap of significant results to file: output_pwys/heatmap.pdf
##  [1] "There are no associations to plot!"
##  2024-09-03 05:23:28.9105 INFO::Writing association plots (one for each significant association) to output folder: output_pwys
##  2024-09-03 05:23:28.914552 INFO::Plotting associations from most to least significant, grouped by metadata
##  2024-09-03 05:23:28.915097 INFO::Plotting data for metadata number 1, antibiotics_current_use
##  2024-09-03 05:23:28.916119 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')

print(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.

17.5 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"