17 Microbe Set Enrichment Analysis (MSEA)
Similar to gene set enrichment analyses for genes (A 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) (Y 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.
A, Subramanian. 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://www.pnas.org/doi/10.1073/pnas.0506580102?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed.
Y, Kou. 2020. “Microbe-Set Enrichment Analysis Facilitates Functional Interpretation of Microbiome Profiling Data.” Scientific Reports 10 (1): 21466. https://www.nature.com/articles/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 A (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 (J 2019). We aim to conduct MSEA analysis based on both taxonomic profiles (species relative abundances) and functional profiles (pathway relative abundances).
J, Lloyd-Price. 2019. “Multi-Omics of the Gut Microbial Ecosystem in Inflammatory Bowel Diseases.” Nature 569 (7758): 655–62.
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 (H 2021). The ranked feature list from the differential abundance analysis serves as an input for the MSEA.
H, Mallick. 2021. “Multivariable Association Discovery in Population-Scale Meta-Omics Studies.” PLOS Computational Biology 17 (11): e1009442. https://journals.plos.org/ploscompbiol/article?id=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"
## 2024-05-03 13:10:52.446823 INFO::Writing function arguments to log file
## 2024-05-03 13:10:52.461587 INFO::Verifying options selected are valid
## 2024-05-03 13:10:52.488531 INFO::Determining format of input files
## 2024-05-03 13:10:52.489274 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-05-03 13:10:52.497612 INFO::Formula for fixed effects: expr ~ disease + age + antibiotics_current_use
## 2024-05-03 13:10:52.498516 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
## 2024-05-03 13:10:52.499309 INFO::Filter data based on min abundance and min prevalence
## 2024-05-03 13:10:52.499879 INFO::Total samples in data: 136
## 2024-05-03 13:10:52.500432 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
## 2024-05-03 13:10:52.509282 INFO::Total filtered features: 452
## 2024-05-03 13:10:52.510074 INFO::Filtered feature names from abundance and prevalence filtering: Clostridiales.bacterium.1_7_47FAA, Enterocloster.asparagiformis, Gordonibacter.pamelaeae, Firmicutes.bacterium.CAG.424, Adlercreutzia.equolifaciens, Anaerotruncus.sp..CAG.528, Parabacteroides.johnsonii, Erysipelothrix.larvae, Proteus.mirabilis, Bacteroides.sp..43_108, Blautia.producta, Tyzzerella.nexilis, Ruminococcus.obeum.CAG.39, X.Clostridium..scindens, Blautia.hydrogenotrophica, Lachnoclostridium.sp..An181, Candidatus.Stoquefichus.sp..KLE1796, Gemmiger.sp..An87, Lachnoclostridium.sp..An138, Harryflintia.acetispora, Streptococcus.thermophilus, Prevotella.buccae, Dorea.sp..CAG.317, Proteus.vulgaris, Proteus.hauseri, X.Clostridium..spiroforme, Blautia.hansenii, Eubacterium.sp..OM08.24, Parabacteroides.sp..CAG.409, Desulfovibrio.piger, Clostridium.sp..CAG.242, Pseudoflavonifractor.sp..An184, Clostridiales.bacterium.CHKCI006, Pseudoflavonifractor.sp..An85, Christensenella.minuta, Bifidobacterium.catenulatum, Slackia.isoflavoniconvertens, Alistipes.inops, Haemophilus.pittmaniae, Amedibacillus.dolichus, Eubacterium.dolichum.CAG.375, Turicibacter.sanguinis, Lactobacillus.delbrueckii, Aeriscardovia.aeriphila, Firmicutes.bacterium.CAG.145, Klebsiella.quasipneumoniae, Cutibacterium.granulosum, Methanobrevibacter.smithii, Porphyromonas.asaccharolytica, Clostridium.perfringens, Clostridium.paraputrificum, Limosilactobacillus.fermentum, Prevotella.sp..CAG.279, Fusobacterium.ulcerans, Bifidobacterium.breve, Prevotella.sp..CAG.891, Lactococcus.piscium, Klebsiella.oxytoca, Citrobacter.freundii, Citrobacter.braakii, Bifidobacterium.animalis, Citrobacter.youngae, Lactobacillus.acidophilus, Anaeroglobus.geminatus, Bifidobacterium.dentium, Clostridium.neonatale, Clostridioides.difficile, Enterococcus.faecium, Morganella.morganii, Streptococcus.oralis, Klebsiella.michiganensis, Clostridium.sp..chh4.2, Bacteroides.nordii, Klebsiella.aerogenes, Leclercia.adecarboxylata, Enterobacter.mori, Kluyvera.ascorbata, Pseudoflavonifractor.capillosus, Ruminococcaceae.bacterium.D5, Raoultella.planticola, Roseburia.sp..CAG.303, Alloscardovia.omnicolens, Eubacterium.sp..CAG.180, Eubacterium.sp..CAG.274, Cutibacterium.acnes, Dialister.sp..CAG.357, Bacteroides.oleiciplenus, Coprobacter.secundus, Kocuria.palustris, Acinetobacter.lwoffii, Micrococcus.lylae, Pararheinheimera.mesophila, Desulfovibrionaceae.bacterium, Massilimicrobiota.timonensis, Fretibacterium.fastidiosum, Streptococcus.vestibularis, Clostridium.sp..CAG.411, Megamonas.funiformis, Megamonas.hypermegale, Megamonas.funiformis.CAG.377, Streptococcus.gordonii, Coprobacillus.cateniformis, Prevotella.colorans, Prevotella.buccalis, Anaerofustis.stercorihominis, Latilactobacillus.sakei, Clostridium.disporicum, Ruminococcus.sp..CAG.330, Streptococcus.agalactiae, Aggregatibacter.segnis, Fusobacterium.nucleatum, Bifidobacterium.pseudolongum, Allisonella.histaminiformans, Prevotella.bivia, Clostridium.sp..CAG.590, Ruminococcus.callidus, Holdemanella.biformis, Escherichia.marmotae, Terrisporobacter.othiniensis, Porphyromonas.somerae, Prevotella.salivae, Citrobacter.amalonaticus, Ligilactobacillus.salivarius, Megasphaera.micronuciformis, Neisseria.sp..oral.taxon.014, Anaerostipes.caccae, Blautia.argi, Escherichia.albertii, Prevotella.disiens, Porphyromonas.uenonis, Peptococcus.niger, Bacteroides.sp..CAG.144, Eubacterium.sp..An11, Blautia.coccoides, Oxalobacter.formigenes, Tractidigestivibacter.scatoligenes, Dialister.pneumosintes, Megasphaera.sp..DISK.18, Clostridium.sp..CAG.167, Hafnia.paralvei, Alistipes.onderdonkii, Coprobacter.sp., Corynebacterium.matruchotii, Bacteroides.stercorirosoris, Staphylococcus.epidermidis, Parabacteroides.goldsteinii, Roseburia.sp..CAG.182, Oscillibacter.sp..PC13, Firmicutes.bacterium.CAG.110, Prevotella.sp..885, Parvimonas.micra, Peptostreptococcus.stomatis, Moraxella.osloensis, Enhydrobacter.aerosaccus, Micrococcus.luteus, Micrococcus.aloeverae, Dialister.micraerophilus, Campylobacter.concisus, Megasphaera.sp..MJR8396C, Cloacibacillus.porcorum, Desulfovibrio.fairfieldensis, Phascolarctobacterium.sp..CAG.266, Rikenella.microfusus, Roseburia.sp..CAG.309, Flavonifractor.sp..An10, Victivallis.vadensis, Firmicutes.bacterium.CAG.95, Enorma.massiliensis, Citrobacter.pasteurii, Streptococcus.salivarius.CAG.79, Veillonella.seminalis, Enterococcus.faecalis, Streptococcus.mitis, Butyricicoccus.pullicaecorum, Prevotella.corporis, Lachnoclostridium.sp..An131, Lachnoclostridium.sp..An118, X.Clostridium..methylpentosum, Haemophilus.parahaemolyticus, Neisseria.flavescens, Veillonella.rogosae, Neisseria.subflava, Sharpea.azabuensis, Anaerofilum.sp..An201, Romboutsia.ilealis, Fusobacterium.periodonticum, Lachnoclostridium.sp..An14, Flavonifractor.sp..An100, Clostridium.sp..CAG.253, Arthrospira.platensis, Lactobacillus.amylovorus, Limosilactobacillus.oris, Streptococcus.lutetiensis, Weissella.cibaria, Rothia.mucilaginosa, Streptococcus.pasteurianus, Prevotella.melaninogenica, Prevotella.jejuni, Veillonella.tobetsuensis, Streptococcus.sp..M334, Actinomyces.sp..ICM47, Streptococcus.sp..A12, Clostridium.sp..CAG.678, Candidatus.Methanomassiliicoccus.intestinalis, Candidatus.Gastranaerophilales.bacterium, Prevotella.denticola, Leuconostoc.lactis, Prevotella.oris, Prevotella.dentalis, X.Eubacterium..infirmum, Shuttleworthia.satelles, Prevotella.nigrescens, Enterococcus.hirae, Adlercreutzia.caecimuris, Citrobacter.portucalensis, Raoultella.ornithinolytica, Dysgonomonas.mossii, Firmicutes.bacterium.CAG.646, Streptococcus.gallolyticus, Christensenella.hongkongensis, Lactobacillus.paragasseri, Lactobacillus.gasseri, Peptostreptococcus.anaerobius, Acinetobacter.ursingii, Lactococcus.lactis, Odoribacter.laneus, Haemophilus.sputorum, Peptoniphilus.harei, Pediococcus.acidilactici, Fusobacterium.gonidiaformans, Fusobacterium.equinum, Leuconostoc.garlicum, Campylobacter.showae, Aeromonas.hydrophila, Aeromonas.dhakensis, Prevotella.stercorea, Phascolarctobacterium.succinatutens, Prevotella.sp..AM42.24, Blastocystis.sp..subtype.1, Bifidobacterium.angulatum, Prevotella.timonensis, Butyrivibrio.crossotus, Limosilactobacillus.vaginalis, Ligilactobacillus.ruminis, Weissella.confusa, Streptococcus.mutans, Clostridium.celatum, Streptococcus.australis, Streptococcus.sanguinis, Schaalia.turicensis, Peptoniphilus.duerdenii, Clostridium.baratii, Streptococcus.sp..F0442, Prevotella.sp..CAG.520, Cronobacter.sakazakii, Actinomyces.urogenitalis, Actinomyces.graevenitzii, Parvimonas.sp..oral.taxon.110, Eikenella.corrodens, Parvimonas.sp..oral.taxon.393, Anaerococcus.vaginalis, Gemella.asaccharolytica, Lachnospiraceae.bacterium.2_1_46FAA, Veillonella.sp..CAG.933, Lacticaseibacillus.rhamnosus, Campylobacter.upsaliensis, Lachnospiraceae.bacterium.oral.taxon.096, Campylobacter.gracilis, Gemmiger.sp..An50, Scardovia.wiggsiae, Limosilactobacillus.reuteri, Enterococcus.avium, Enterococcus.thailandicus, Paucilactobacillus.vaccinostercus, Enterococcus.casseliflavus, Bifidobacterium.pullorum, Actinomyces.sp..oral.taxon.180, Enterococcus.sp..3H8_DIV0648, Enterococcus.gallinarum, Varibaculum.cambriense, Veillonella.rodentium, Lactobacillus.crispatus, Lawsonella.clevelandensis, Porphyromonas.sp..HMSC065F10, Citrobacter.werkmanii, Lactobacillus.johnsonii, Sarcina.ventriculi, Streptococcus.macedonicus, Klebsiella.variicola.CAG.634, Phytobacter.palmae, Enterococcus.raffinosus, Prevotella.amnii, Catenibacterium.mitsuokai, Saccharomyces.cerevisiae, Eubacterium.limosum, Staphylococcus.hominis, Alloprevotella.tannerae, Haemophilus.paraphrohaemolyticus, X.Eubacterium..brachy, Ruminococcus.sp..CAG.403, Fannyhessea.vaginae, Cronobacter.malonaticus, Streptococcus.infantis, Actinomyces.sp..oral.taxon.181, Clostridium.butyricum, Abiotrophia.sp..HMSC24B09, Gemella.haemolysans, Firmicutes.bacterium.CAG.170, Solobacterium.moorei, Pseudomonas.guguanensis, Lactobacillus.jensenii, Clostridium.cadaveris, Aggregatibacter.sp..oral.taxon.458, Anaerocolumna.aminovalerica, Clostridium.sporogenes, Clostridium.botulinum, Campylobacter.hominis, Murimonas.intestini, Clostridium.sp..CAG.964, Pediococcus.pentosaceus, Corynebacterium.amycolatum, Parvimonas.sp..KA00067, Bacteroidales.bacterium.KA00251, Fusobacterium.mortiferum, Lacrimispora.celerecrescens, Butyrivibrio.sp..CAG.318, Cellulosilyticum.lentocellum, Eubacteriaceae.bacterium.CHKCI005, Eubacterium.coprostanoligenes, Firmicutes.bacterium.CAG.238, Dysgonomonas.gadei, Bacteroides.fluxus, Bacteroides.sp..D2, Citrobacter.farmeri, Gemella.sanguinis, Sanguibacteroides.justesenii, Porphyromonas.endodontalis, Streptococcus.peroris, Actinomyces.sp..HPA0247, Lactiplantibacillus.plantarum, Limosilactobacillus.mucosae, Providencia.alcalifaciens, Robinsoniella.sp..RHS, Fructilactobacillus.sanfranciscensis, Prevotella.sp..CAG.1185, Peptoniphilus.lacrimalis, Gemella.morbillorum, Frigoribacterium.sp..Leaf8, Clostridium.sp..MSTE9, X.Clostridium..hylemonae, Treponema.lecithinolyticum, Faecalicoccus.pleomorphus, Flavonifractor.sp..An306, Prevotella.oralis, Peptoniphilus.sp..BV3C26, Brachyspira.pilosicoli, Bacteroides.sp..OM08.11, Proteus.penneri, Corynebacterium.kroppenstedtii, Faecalitalea.cylindroides, Wohlfahrtiimonas.chitiniclastica, Bacteroides.clarus, Acidaminococcus.sp..CAG.542, Peptoniphilus.coxii, Alistipes.timonensis, Clostridium.sp..D5, Prevotella.bergensis, Ruminococcus.sp..CAG.563, Kluyvera.cryocrescens, Ezakiella.coagulans, X.Bacteroides..pectinophilus, Bacteroides.sp..CAG.661, Bacteroides.sp..CAG.598, Comamonas.kerstersii, Firmicutes.bacterium.CAG.534, Clostridium.sp..CAG.413, Schaalia.odontolytica, Streptococcus.cristatus, Actinomyces.sp..HMSC035G02, Lancefieldella.rimae, Streptococcus.milleri, Finegoldia.magna, Lancefieldella.parvula, Prevotella.oulorum, Dellaglioa.algida, Lactonifactor.longoviformis, Hafnia.alvei, Neisseria.cinerea, Corynebacterium.oculi, Ruminococcus.sp..CAG.488, Corynebacterium.accolens, Prevotella.sp..S7.1.8, Campylobacter.ureolyticus, Atopobium.deltae, Gleimia.europaea, Mitsuokella.jalaludinii, Mitsuokella.multacida, Peptoniphilus.sp..HMSC062D09, Anaerococcus.lactolyticus, Granulicatella.adiacens, Anaerosporobacter.mobilis, Clostridium.sp..7_2_43FAA, Kosakonia.sacchari, Staphylococcus.aureus, Ligilactobacillus.animalis, Weissella.viridescens, Dysgonomonas.sp..37.18, Serratia.liquefaciens, Streptococcus.sp..HPH0090, Anaerostipes.sp..494a, Lactococcus.petauri, Obesumbacterium.proteus, Fusobacterium.sp..oral.taxon.370, X.Butyribacterium..methylotrophicum, Phocaeicola.sartorii, Bacteroides.sp..CAG.530, Megasphaera.elsdenii, Prevotella.sp..CAG.1092, Fusobacterium.sp..CAG.439, Anaeromassilibacillus.sp..An172, Aggregatibacter.aphrophilus, Aerococcus.urinaeequi, Pseudomonas.fragi, Thermoleophilum.album, Bacteroides.sp..CAG.633, Serratia.marcescens, Actinotignum.timonense, Fusobacterium.naviforme, Haemophilus.influenzae, Prevotella.intermedia, Bacteroidetes.oral.taxon.274, Citrobacter.europaeus, Bifidobacterium.asteroides, Enterococcus.durans, Enterococcus.pseudoavium, Microvirgula.aerodenitrificans, Yersinia.frederiksenii, Faecalicatena.orotica, Enterococcus.asini, Paenibacillus.macerans, Bavariicoccus.seileri, Kluyvera.georgiana, Prevotella.histicola, Prevotella.pallens, Chlamydia.ibidis, Enterococcus.mundtii, Anaerostipes.sp..992a, Actinobaculum.sp..oral.taxon.183, Lachnoclostridium.sp..An298, Haemophilus.haemolyticus, Enterococcus.dispar, Atopobium.minutum
## 2024-05-03 13:10:52.512732 INFO::Total filtered features with variance filtering: 0
## 2024-05-03 13:10:52.513243 INFO::Filtered feature names from variance filtering:
## 2024-05-03 13:10:52.513662 INFO::Running selected normalization method: NONE
## 2024-05-03 13:10:52.514127 INFO::Applying z-score to standardize continuous metadata
## 2024-05-03 13:10:52.518321 INFO::Running selected transform method: LOG
## 2024-05-03 13:10:52.521229 INFO::Running selected analysis method: LM
## 2024-05-03 13:10:52.524762 INFO::Fitting model to feature number 1, Phocaeicola.vulgatus
## 2024-05-03 13:10:52.53113 INFO::Fitting model to feature number 2, Bacteroides.uniformis
## 2024-05-03 13:10:52.533404 INFO::Fitting model to feature number 3, Bacteroides.thetaiotaomicron
## 2024-05-03 13:10:52.535681 INFO::Fitting model to feature number 4, Faecalibacterium.prausnitzii
## 2024-05-03 13:10:52.537966 INFO::Fitting model to feature number 5, Roseburia.faecis
## 2024-05-03 13:10:52.540174 INFO::Fitting model to feature number 6, Bacteroides.caccae
## 2024-05-03 13:10:52.542436 INFO::Fitting model to feature number 7, Enterocloster.clostridioformis
## 2024-05-03 13:10:52.544674 INFO::Fitting model to feature number 8, Bacteroides.fragilis
## 2024-05-03 13:10:52.546966 INFO::Fitting model to feature number 9, Fusicatenibacter.saccharivorans
## 2024-05-03 13:10:52.549402 INFO::Fitting model to feature number 10, Flavonifractor.plautii
## 2024-05-03 13:10:52.551659 INFO::Fitting model to feature number 11, Dialister.invisus
## 2024-05-03 13:10:52.553897 INFO::Fitting model to feature number 12, Ruminococcus.bicirculans
## 2024-05-03 13:10:52.5561 INFO::Fitting model to feature number 13, Blautia.sp..CAG.257
## 2024-05-03 13:10:52.558378 INFO::Fitting model to feature number 14, X.Ruminococcus..gnavus
## 2024-05-03 13:10:52.560596 INFO::Fitting model to feature number 15, Dorea.longicatena
## 2024-05-03 13:10:52.562842 INFO::Fitting model to feature number 16, Anaerobutyricum.hallii
## 2024-05-03 13:10:52.565072 INFO::Fitting model to feature number 17, Enterocloster.bolteae
## 2024-05-03 13:10:52.567287 INFO::Fitting model to feature number 18, Eubacterium.sp..CAG.38
## 2024-05-03 13:10:52.569521 INFO::Fitting model to feature number 19, Lacrimispora.saccharolytica
## 2024-05-03 13:10:52.57174 INFO::Fitting model to feature number 20, Intestinimonas.butyriciproducens
## 2024-05-03 13:10:52.573979 INFO::Fitting model to feature number 21, Anaerostipes.hadrus
## 2024-05-03 13:10:52.576178 INFO::Fitting model to feature number 22, Anaerotignum.lactatifermentans
## 2024-05-03 13:10:52.578418 INFO::Fitting model to feature number 23, Ruthenibacterium.lactatiformans
## 2024-05-03 13:10:52.580688 INFO::Fitting model to feature number 24, Lawsonibacter.asaccharolyticus
## 2024-05-03 13:10:52.582933 INFO::Fitting model to feature number 25, Clostridium.bolteae.CAG.59
## 2024-05-03 13:10:52.585176 INFO::Fitting model to feature number 26, X.Clostridium..symbiosum
## 2024-05-03 13:10:52.587384 INFO::Fitting model to feature number 27, Anaerotruncus.colihominis
## 2024-05-03 13:10:52.58964 INFO::Fitting model to feature number 28, Agathobaculum.butyriciproducens
## 2024-05-03 13:10:52.591941 INFO::Fitting model to feature number 29, Escherichia.coli
## 2024-05-03 13:10:52.594243 INFO::Fitting model to feature number 30, Eisenbergiella.massiliensis
## 2024-05-03 13:10:52.596445 INFO::Fitting model to feature number 31, Enterocloster.citroniae
## 2024-05-03 13:10:52.599012 INFO::Fitting model to feature number 32, Bacteroides.stercoris
## 2024-05-03 13:10:52.601408 INFO::Fitting model to feature number 33, Monoglobus.pectinilyticus
## 2024-05-03 13:10:52.603823 INFO::Fitting model to feature number 34, Alistipes.putredinis
## 2024-05-03 13:10:52.606087 INFO::Fitting model to feature number 35, Parabacteroides.distasonis
## 2024-05-03 13:10:52.608324 INFO::Fitting model to feature number 36, Roseburia.hominis
## 2024-05-03 13:10:52.610595 INFO::Fitting model to feature number 37, Bifidobacterium.longum
## 2024-05-03 13:10:52.612831 INFO::Fitting model to feature number 38, X.Eubacterium..siraeum
## 2024-05-03 13:10:52.61508 INFO::Fitting model to feature number 39, Parabacteroides.merdae
## 2024-05-03 13:10:52.617323 INFO::Fitting model to feature number 40, Roseburia.inulinivorans
## 2024-05-03 13:10:52.619547 INFO::Fitting model to feature number 41, Bacteroides.cellulosilyticus
## 2024-05-03 13:10:52.621818 INFO::Fitting model to feature number 42, Bacteroides.ovatus
## 2024-05-03 13:10:52.624048 INFO::Fitting model to feature number 43, Blautia.wexlerae
## 2024-05-03 13:10:52.626307 INFO::Fitting model to feature number 44, Bifidobacterium.adolescentis
## 2024-05-03 13:10:52.628524 INFO::Fitting model to feature number 45, Lachnospira.eligens
## 2024-05-03 13:10:52.630792 INFO::Fitting model to feature number 46, X.Clostridium..leptum
## 2024-05-03 13:10:52.633029 INFO::Fitting model to feature number 47, Eggerthella.lenta
## 2024-05-03 13:10:52.635255 INFO::Fitting model to feature number 48, Bacteroides.xylanisolvens
## 2024-05-03 13:10:52.637501 INFO::Fitting model to feature number 49, Phocaeicola.dorei
## 2024-05-03 13:10:52.639716 INFO::Fitting model to feature number 50, Enterocloster.lavalensis
## 2024-05-03 13:10:52.641947 INFO::Fitting model to feature number 51, X.Eubacterium..rectale
## 2024-05-03 13:10:52.644172 INFO::Fitting model to feature number 52, Akkermansia.muciniphila
## 2024-05-03 13:10:52.646411 INFO::Fitting model to feature number 53, Acidaminococcus.intestini
## 2024-05-03 13:10:52.648616 INFO::Fitting model to feature number 54, X.Ruminococcus..torques
## 2024-05-03 13:10:52.650857 INFO::Fitting model to feature number 55, Bacteroides.salyersiae
## 2024-05-03 13:10:52.653095 INFO::Fitting model to feature number 56, Sellimonas.intestinalis
## 2024-05-03 13:10:52.65536 INFO::Fitting model to feature number 57, Bacteroides.faecis
## 2024-05-03 13:10:52.657607 INFO::Fitting model to feature number 58, Roseburia.intestinalis
## 2024-05-03 13:10:52.659816 INFO::Fitting model to feature number 59, Collinsella.aerofaciens
## 2024-05-03 13:10:52.662077 INFO::Fitting model to feature number 60, Bacteroides.intestinalis
## 2024-05-03 13:10:52.664345 INFO::Fitting model to feature number 61, Bacteroides.faecis.CAG.32
## 2024-05-03 13:10:52.666612 INFO::Fitting model to feature number 62, X.Ruminococcus..lactaris
## 2024-05-03 13:10:52.668847 INFO::Fitting model to feature number 63, Prevotella.copri
## 2024-05-03 13:10:52.671127 INFO::Fitting model to feature number 64, Eisenbergiella.tayi
## 2024-05-03 13:10:52.673409 INFO::Fitting model to feature number 65, Klebsiella.pneumoniae
## 2024-05-03 13:10:52.675647 INFO::Fitting model to feature number 66, Intestinibacter.bartlettii
## 2024-05-03 13:10:52.677914 INFO::Fitting model to feature number 67, Erysipelatoclostridium.ramosum
## 2024-05-03 13:10:52.680171 INFO::Fitting model to feature number 68, Barnesiella.intestinihominis
## 2024-05-03 13:10:52.682438 INFO::Fitting model to feature number 69, Blautia.obeum
## 2024-05-03 13:10:52.684681 INFO::Fitting model to feature number 70, Bacteroides.finegoldii
## 2024-05-03 13:10:52.686936 INFO::Fitting model to feature number 71, Parasutterella.excrementihominis
## 2024-05-03 13:10:52.689215 INFO::Fitting model to feature number 72, Phascolarctobacterium.faecium
## 2024-05-03 13:10:52.691462 INFO::Fitting model to feature number 73, Lachnospira.pectinoschiza
## 2024-05-03 13:10:52.693738 INFO::Fitting model to feature number 74, Clostridium.sp..CAG.58
## 2024-05-03 13:10:52.695999 INFO::Fitting model to feature number 75, Bilophila.wadsworthia
## 2024-05-03 13:10:52.698262 INFO::Fitting model to feature number 76, Turicimonas.muris
## 2024-05-03 13:10:52.700534 INFO::Fitting model to feature number 77, Proteobacteria.bacterium.CAG.139
## 2024-05-03 13:10:52.7028 INFO::Fitting model to feature number 78, Anaeromassilibacillus.sp..An250
## 2024-05-03 13:10:52.705047 INFO::Fitting model to feature number 79, Hungatella.hathewayi
## 2024-05-03 13:10:52.70729 INFO::Fitting model to feature number 80, Alistipes.finegoldii
## 2024-05-03 13:10:52.709574 INFO::Fitting model to feature number 81, Ruminococcus.bromii
## 2024-05-03 13:10:52.711781 INFO::Fitting model to feature number 82, Odoribacter.splanchnicus
## 2024-05-03 13:10:52.714023 INFO::Fitting model to feature number 83, Butyricimonas.virosa
## 2024-05-03 13:10:52.716238 INFO::Fitting model to feature number 84, Oscillibacter.sp..CAG.241
## 2024-05-03 13:10:52.718507 INFO::Fitting model to feature number 85, Alistipes.indistinctus
## 2024-05-03 13:10:52.720773 INFO::Fitting model to feature number 86, Coprococcus.comes
## 2024-05-03 13:10:52.723064 INFO::Fitting model to feature number 87, Gemmiger.formicilis
## 2024-05-03 13:10:52.725314 INFO::Fitting model to feature number 88, Holdemania.filiformis
## 2024-05-03 13:10:52.727544 INFO::Fitting model to feature number 89, Firmicutes.bacterium.CAG.83
## 2024-05-03 13:10:52.729777 INFO::Fitting model to feature number 90, Dorea.formicigenerans
## 2024-05-03 13:10:52.732005 INFO::Fitting model to feature number 91, Collinsella.intestinalis
## 2024-05-03 13:10:52.734235 INFO::Fitting model to feature number 92, Oscillibacter.sp..57_20
## 2024-05-03 13:10:52.736465 INFO::Fitting model to feature number 93, Firmicutes.bacterium.CAG.94
## 2024-05-03 13:10:52.738749 INFO::Fitting model to feature number 94, Dielma.fastidiosa
## 2024-05-03 13:10:52.740997 INFO::Fitting model to feature number 95, Roseburia.sp..CAG.471
## 2024-05-03 13:10:52.743228 INFO::Fitting model to feature number 96, X.Clostridium..innocuum
## 2024-05-03 13:10:52.745493 INFO::Fitting model to feature number 97, Haemophilus.parainfluenzae
## 2024-05-03 13:10:52.747725 INFO::Fitting model to feature number 98, Veillonella.dispar
## 2024-05-03 13:10:52.750035 INFO::Fitting model to feature number 99, Veillonella.parvula
## 2024-05-03 13:10:52.752276 INFO::Fitting model to feature number 100, Veillonella.infantium
## 2024-05-03 13:10:52.754755 INFO::Fitting model to feature number 101, Streptococcus.salivarius
## 2024-05-03 13:10:52.756986 INFO::Fitting model to feature number 102, Enterocloster.aldenensis
## 2024-05-03 13:10:52.759259 INFO::Fitting model to feature number 103, Veillonella.atypica
## 2024-05-03 13:10:52.761513 INFO::Fitting model to feature number 104, Phocaeicola.plebeius
## 2024-05-03 13:10:52.763731 INFO::Fitting model to feature number 105, Paraprevotella.xylaniphila
## 2024-05-03 13:10:52.766032 INFO::Fitting model to feature number 106, Bacteroides.eggerthii
## 2024-05-03 13:10:52.768343 INFO::Fitting model to feature number 107, Alistipes.shahii
## 2024-05-03 13:10:52.770687 INFO::Fitting model to feature number 108, Butyricimonas.synergistica
## 2024-05-03 13:10:52.772958 INFO::Fitting model to feature number 109, Haemophilus.sp..HMSC71H05
## 2024-05-03 13:10:52.775196 INFO::Fitting model to feature number 110, Eubacterium.ramulus
## 2024-05-03 13:10:52.777485 INFO::Fitting model to feature number 111, Coprobacter.fastidiosus
## 2024-05-03 13:10:52.779742 INFO::Fitting model to feature number 112, Lactobacillus.rogosae
## 2024-05-03 13:10:52.781999 INFO::Fitting model to feature number 113, Phocaeicola.massiliensis
## 2024-05-03 13:10:52.784266 INFO::Fitting model to feature number 114, Streptococcus.parasanguinis
## 2024-05-03 13:10:52.786536 INFO::Fitting model to feature number 115, Ruminococcaceae.bacterium.D16
## 2024-05-03 13:10:52.788749 INFO::Fitting model to feature number 116, Veillonella.sp..T11011.6
## 2024-05-03 13:10:52.791003 INFO::Fitting model to feature number 117, Klebsiella.variicola
## 2024-05-03 13:10:52.793266 INFO::Fitting model to feature number 118, Bacteroides.galacturonicus
## 2024-05-03 13:10:52.795514 INFO::Fitting model to feature number 119, Clostridium.sp..CAG.299
## 2024-05-03 13:10:52.79776 INFO::Fitting model to feature number 120, Eubacterium.ventriosum
## 2024-05-03 13:10:52.799999 INFO::Fitting model to feature number 121, Bifidobacterium.bifidum
## 2024-05-03 13:10:52.802284 INFO::Fitting model to feature number 122, Bifidobacterium.pseudocatenulatum
## 2024-05-03 13:10:52.804526 INFO::Fitting model to feature number 123, Coprococcus.eutactus
## 2024-05-03 13:10:52.806791 INFO::Fitting model to feature number 124, Eubacterium.sp..CAG.251
## 2024-05-03 13:10:52.809064 INFO::Fitting model to feature number 125, Phocaeicola.coprocola
## 2024-05-03 13:10:52.811281 INFO::Fitting model to feature number 126, Paraprevotella.clara
## 2024-05-03 13:10:52.813525 INFO::Fitting model to feature number 127, Coprococcus.catus
## 2024-05-03 13:10:52.833271 INFO::Counting total values for each feature
## 2024-05-03 13:10:52.844642 INFO::Writing filtered data to file output_species/features/filtered_data.tsv
## 2024-05-03 13:10:52.857105 INFO::Writing filtered, normalized data to file output_species/features/filtered_data_norm.tsv
## 2024-05-03 13:10:52.869141 INFO::Writing filtered, normalized, transformed data to file output_species/features/filtered_data_norm_transformed.tsv
## 2024-05-03 13:10:52.886435 INFO::Writing residuals to file output_species/fits/residuals.rds
## 2024-05-03 13:10:52.892456 INFO::Writing fitted values to file output_species/fits/fitted.rds
## 2024-05-03 13:10:52.89774 INFO::Writing all results to file (ordered by increasing q-values): output_species/all_results.tsv
## 2024-05-03 13:10:52.900541 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
## 2024-05-03 13:10:52.901392 INFO::Writing heatmap of significant results to file: output_species/heatmap.pdf
## 2024-05-03 13:10:53.005846 INFO::Writing association plots (one for each significant association) to output folder: output_species
## 2024-05-03 13:10:53.008474 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-05-03 13:10:53.009035 INFO::Plotting data for metadata number 1, age
## 2024-05-03 13:10:53.009916 INFO::Creating scatter plot for continuous data, age vs Alistipes.indistinctus
## 2024-05-03 13:10:53.191194 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bicirculans
## 2024-05-03 13:10:53.410967 INFO::Creating scatter plot for continuous data, age vs Lacrimispora.saccharolytica
## 2024-05-03 13:10:53.616722 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
## 2024-05-03 13:10:53.739671 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
## 2024-05-03 13:10:53.861216 INFO::Creating scatter plot for continuous data, age vs X.Ruminococcus..gnavus
## 2024-05-03 13:10:53.982362 INFO::Creating scatter plot for continuous data, age vs Lachnospira.eligens
## 2024-05-03 13:10:54.10379 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
## 2024-05-03 13:10:54.234546 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.virosa
## 2024-05-03 13:10:54.361525 INFO::Creating scatter plot for continuous data, age vs Lawsonibacter.asaccharolyticus
## 2024-05-03 13:10:54.500665 INFO::Creating scatter plot for continuous data, age vs X.Eubacterium..siraeum
## 2024-05-03 13:10:54.622384 INFO::Creating scatter plot for continuous data, age vs Butyricimonas.synergistica
## 2024-05-03 13:10:54.74414 INFO::Creating scatter plot for continuous data, age vs Monoglobus.pectinilyticus
## 2024-05-03 13:10:54.865636 INFO::Creating scatter plot for continuous data, age vs Veillonella.atypica
## 2024-05-03 13:10:57.152792 INFO::Plotting data for metadata number 2, disease
## 2024-05-03 13:10:57.153991 INFO::Creating boxplot for categorical data, disease vs Alistipes.putredinis
## 2024-05-03 13:10:57.300863 INFO::Creating boxplot for categorical data, disease vs Gemmiger.formicilis
## 2024-05-03 13:10:57.422965 INFO::Creating boxplot for categorical data, disease vs X.Ruminococcus..torques
## 2024-05-03 13:10:57.533556 INFO::Creating boxplot for categorical data, disease vs Ruminococcus.bicirculans
## 2024-05-03 13:10:57.650697 INFO::Creating boxplot for categorical data, disease vs Sellimonas.intestinalis
## 2024-05-03 13:10:57.759497 INFO::Creating boxplot for categorical data, disease vs X.Clostridium..leptum
## 2024-05-03 13:10:57.868272 INFO::Creating boxplot for categorical data, disease vs Alistipes.shahii
## 2024-05-03 13:10:58.915357 INFO::Plotting data for metadata number 3, antibiotics_current_use
## 2024-05-03 13:10:58.916558 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Coprobacter.fastidiosus
## 2024-05-03 13:10:59.027047 INFO::Creating boxplot for categorical data, antibiotics_current_use vs X.Eubacterium..rectale
## 2024-05-03 13:10:59.149286 INFO::Creating boxplot for categorical data, antibiotics_current_use vs Agathobaculum.butyriciproducensUnlike gene expression studies, we do not have well-defined signatures or modules for microbiome data. Here, we will construct data-driven modules using weighted gene co-expression network analysis (WGCNA) L (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.
L, Geistlinger. 2023. “BugSigDB Captures Patterns of Differential Abundance Across a Broad Range of Host-Associated Microbial Signatures.” Nature Biotechnology. https://doi.org/10.1038/s41587-023-01872-y.
library(WGCNA)
datExpr <- as.data.frame(t(fit_data$residuals))
gsg = goodSamplesGenes(datExpr, verbose = 3)
## Flagging genes and samples with too many missing values...
## ..step 1
## ..Excluding 6 samples from the calculation due to too many missing genes.
## ..step 2
gsg$allOK
## [1] FALSEIf the last statement returns TRUE, no outliers are identified. If not, we need to remove the outliers from the data.
if (!gsg$allOK)
{if (sum(!gsg$goodGenes) > 0)
printFlush(paste("Removing genes:", paste(names(datExpr)[!gsg$goodGenes], collapse = ", ")));
if (sum(!gsg$goodSamples) > 0)
printFlush(paste("Removing samples:", paste(rownames(datExpr)[!gsg$goodSamples], collapse =", ")))
datExpr = datExpr[gsg$goodSamples, gsg$goodGenes]
}
## Removing samples: CSM5MCVB_P, CSM79HNY_P, ESM5GEYY_P, ESM718U9_P, MSM6J2N6_P, MSM9VZLX_PAfter removing the outliers, we need to choose a suitable soft threshold parameter for creating the modules as part of the WGCNA algorithm. This power value must produce a graph similar to a scale-free network. We can use the mean connectivity graphic for the selection of this power parameter.
# Choose a set of soft threshold parameters
powers = c(c(1:20), seq(from = 22, to=30, by=2))
sft = pickSoftThreshold(datExpr, powerVector = powers, verbose = 5, dataIsExpr = TRUE, RsquaredCut = 0.30)
## pickSoftThreshold: will use block size 127.
## pickSoftThreshold: calculating connectivity for given powers...
## ..working on genes 1 through 127 of 127
## Power SFT.R.sq slope truncated.R.sq mean.k. median.k. max.k.
## 1 1 0.1790 -0.972 0.85700 15.00000 1.48e+01 23.0000
## 2 2 0.3450 -0.729 0.63500 2.95000 2.85e+00 5.6900
## 3 3 0.2400 -4.130 0.12700 0.80400 7.36e-01 2.6100
## 4 4 0.3100 -4.870 0.19900 0.29200 2.14e-01 1.6700
## 5 5 0.2150 -4.030 -0.00880 0.13700 7.02e-02 1.2200
## 6 6 0.1800 -2.930 -0.05130 0.07910 2.46e-02 0.9570
## 7 7 0.1380 -2.240 -0.10100 0.05320 9.33e-03 0.7900
## 8 8 0.2200 -2.790 -0.00259 0.03940 3.71e-03 0.6690
## 9 9 0.0872 -1.450 -0.09130 0.03100 1.50e-03 0.5760
## 10 10 0.1190 -1.630 -0.07660 0.02540 6.73e-04 0.5020
## 11 11 0.0562 -1.250 -0.03470 0.02130 2.66e-04 0.4410
## 12 12 0.1620 -2.150 -0.07720 0.01810 1.12e-04 0.3910
## 13 13 0.2120 -2.350 -0.00949 0.01560 4.84e-05 0.3480
## 14 14 0.2480 -2.410 0.04950 0.01360 2.12e-05 0.3110
## 15 15 0.2890 -2.470 0.15200 0.01190 9.53e-06 0.2810
## 16 16 0.3030 -2.420 0.18400 0.01050 4.33e-06 0.2580
## 17 17 0.2410 -2.380 0.02450 0.00933 1.98e-06 0.2380
## 18 18 0.3190 -2.640 0.15400 0.00830 9.03e-07 0.2180
## 19 19 0.3320 -2.600 0.17700 0.00741 4.03e-07 0.2010
## 20 20 0.2370 -2.050 0.02180 0.00664 1.70e-07 0.1840
## 21 22 0.2350 -2.000 0.07520 0.00536 3.40e-08 0.1560
## 22 24 0.2430 -2.230 0.09560 0.00437 6.98e-09 0.1310
## 23 26 0.2370 -2.120 0.26200 0.00358 1.44e-09 0.1110
## 24 28 0.2490 -2.080 0.26200 0.00294 2.93e-10 0.0937
## 25 30 0.2490 -2.290 0.30100 0.00243 5.67e-11 0.0791In this step, we will conduct a one-step module detection based on the selected soft threshold parameter selected above.
power = sft$powerEstimate
net = blockwiseModules(datExpr,
power = power,
corFnc="bicor",
corOptions=list(maxPOutliers=0.1),
networkType ="unsigned",
maxBlockSize = ncol(datExpr),
minModuleSize = 3,
TOMType = "unsigned",
reassignThreshold = 0,
mergeCutHeight = 0,
verbose = 3)
## Calculating module eigengenes block-wise from all genes
## Flagging genes and samples with too many missing values...
## ..step 1
## ..Working on block 1 .
## TOM calculation: adjacency..
## ..will not use multithreading.
## Fraction of slow calculations: 0.000000
## ..connectivity..
## ..matrix multiplication (system BLAS)..
## ..normalization..
## ..done.
## ....clustering..
## ....detecting modules..
## ....calculating module eigengenes..
## ....checking kME in modules..
## ..removing 3 genes from module 1 because their KME is too low.
## ..removing 3 genes from module 2 because their KME is too low.
## ..removing 2 genes from module 3 because their KME is too low.
## ..removing 1 genes from module 5 because their KME is too low.
## ..removing 1 genes from module 7 because their KME is too low.
## ..merging modules that are too close..
## mergeCloseModules: Merging modules whose distance is less than 0
## Calculating new MEs...
####################
# How many modules #
####################
ncol(net$MEs)
## [1] 14
table(net$colors)
##
## black blue brown green greenyellow grey
## 7 14 13 12 5 10
## magenta pink purple red salmon tan
## 6 6 5 11 3 5
## turquoise yellow
## 18 12The WGCNA algorithm produced 14 modules which we can visualize as follows.
##########################
# Plot module dendrogram #
##########################
eigenGenes <- net$MEs
MEDiss = 1-cor(eigenGenes)
METree = hclust(as.dist(MEDiss), method = "average")
plot(METree, main = "Clustering of module eigengenes", xlab = "", sub = "")
Next, we calculate hub genes for the modules and create the mapping files to proceed with the MSEA.
###########################################
# Re-calculate modules and find hub genes #
###########################################
moduleColors <- net$colors
MEs0 = moduleEigengenes(datExpr, moduleColors)$eigengenes
modules_data = orderMEs(MEs0)
#######################
# Create mapping file #
#######################
library(tidyverse)
feature_by_modules <- as.data.frame(net$colors)
feature_by_modules <- rownames_to_column(feature_by_modules)
colnames(feature_by_modules) <- c('Feature', 'Module')
features_mapping <- feature_by_modules
features_mapping$Module <- paste('ME', features_mapping$Module, sep = '')Finally, we will run the MSEA analysis on the modules we constructed using WGCNA. Here, we first create a wrapper for the MSEA analysis using the gsEasy package.
library(reshape2)
library(gsEasy)
################
# MSEA Wrapper #
################
run_MSEA <- function(microbeSet, # A list
ranked_features, # Ranked list of featured
filter.count = 3,
seed = 1234,
fdr.correction = 'BH') {
###################
# Filter out sets #
##################
microbeSet0 <- microbeSet
cond <- sapply(microbeSet0, function(x) length(x) > filter.count)
microbeSet <- microbeSet0[cond]
lengthmicrobeSet <- as.data.frame(reshape2::melt(lapply(microbeSet, function(x) length(x))))
colnames(lengthmicrobeSet) <- c('Freq','Set')
################
# Classic MSEA #
################
set.seed(seed)
enrichment <- as.data.frame(sapply(microbeSet, function(set) gset(S = set, r = ranked_features)))
colnames(enrichment)<-'ES'
enrichment <- rownames_to_column(enrichment, 'Set')
enrichment <- merge(enrichment, lengthmicrobeSet, 'Set')
enrichment$qval <- p.adjust(enrichment$ES, fdr.correction)
##########
# Return #
##########
return(enrichment)
}Before running the MSEA, we also need to rank the differential analysis results from MaAsLin2. We use the topGo package to create a list of microbe sets from the mapping file created above.
###################
# Rank DA results #
###################
results <- fit_data$results %>% filter(metadata == 'disease')
results$qval <- p.adjust(results$pval, 'BH')
results <- results[order(results$qval, decreasing = FALSE), ]
###################
# MSEA Processing #
###################
library(topGO)
module_map <- features_mapping
mod.gs <- tapply(module_map$Module, module_map$Feature, as.character)
microbeSet <- inverseList(mod.gs)
microbeSet
## $MEblack
## [1] "Anaeromassilibacillus.sp..An250" "Anaerotruncus.colihominis"
## [3] "Blautia.wexlerae" "Eisenbergiella.tayi"
## [5] "Firmicutes.bacterium.CAG.94" "Ruthenibacterium.lactatiformans"
## [7] "Sellimonas.intestinalis"
##
## $MEblue
## [1] "Clostridium.sp..CAG.58" "Erysipelatoclostridium.ramosum"
## [3] "Haemophilus.parainfluenzae" "Intestinibacter.bartlettii"
## [5] "Klebsiella.pneumoniae" "Klebsiella.variicola"
## [7] "Lawsonibacter.asaccharolyticus" "Streptococcus.parasanguinis"
## [9] "Streptococcus.salivarius" "Veillonella.atypica"
## [11] "Veillonella.dispar" "Veillonella.infantium"
## [13] "Veillonella.parvula" "Veillonella.sp..T11011.6"
##
## $MEbrown
## [1] "Alistipes.finegoldii" "Alistipes.indistinctus"
## [3] "Alistipes.putredinis" "Alistipes.shahii"
## [5] "Bacteroides.xylanisolvens" "Bilophila.wadsworthia"
## [7] "Firmicutes.bacterium.CAG.83" "Odoribacter.splanchnicus"
## [9] "Oscillibacter.sp..57_20" "Oscillibacter.sp..CAG.241"
## [11] "Phocaeicola.dorei" "Ruminococcus.bromii"
## [13] "X.Eubacterium..siraeum"
##
## $MEgreen
## [1] "Blautia.sp..CAG.257" "Clostridium.bolteae.CAG.59"
## [3] "Eggerthella.lenta" "Eisenbergiella.massiliensis"
## [5] "Enterocloster.aldenensis" "Enterocloster.bolteae"
## [7] "Enterocloster.citroniae" "Enterocloster.clostridioformis"
## [9] "Enterocloster.lavalensis" "Flavonifractor.plautii"
## [11] "Lacrimispora.saccharolytica" "X.Clostridium..symbiosum"
##
## $MEgreenyellow
## [1] "Coprobacter.fastidiosus" "Escherichia.coli"
## [3] "Hungatella.hathewayi" "X.Clostridium..innocuum"
## [5] "X.Ruminococcus..gnavus"
##
## $MEgrey
## [1] "Anaerotignum.lactatifermentans" "Bacteroides.cellulosilyticus"
## [3] "Bacteroides.fragilis" "Collinsella.intestinalis"
## [5] "Eubacterium.sp..CAG.38" "Haemophilus.sp..HMSC71H05"
## [7] "Intestinimonas.butyriciproducens" "Lachnospira.eligens"
## [9] "Roseburia.intestinalis" "Roseburia.sp..CAG.471"
##
## $MEmagenta
## [1] "Bacteroides.finegoldii" "Paraprevotella.clara"
## [3] "Paraprevotella.xylaniphila" "Phocaeicola.coprocola"
## [5] "Phocaeicola.plebeius" "Prevotella.copri"
##
## $MEpink
## [1] "Bacteroides.galacturonicus" "Bifidobacterium.pseudocatenulatum"
## [3] "Eubacterium.sp..CAG.251" "Lachnospira.pectinoschiza"
## [5] "Lactobacillus.rogosae" "Phocaeicola.massiliensis"
##
## $MEpurple
## [1] "Barnesiella.intestinihominis" "Butyricimonas.synergistica"
## [3] "Butyricimonas.virosa" "Coprococcus.eutactus"
## [5] "Ruminococcus.bicirculans"
##
## $MEred
## [1] "Akkermansia.muciniphila" "Bacteroides.intestinalis"
## [3] "Clostridium.sp..CAG.299" "Dialister.invisus"
## [5] "Dielma.fastidiosa" "Holdemania.filiformis"
## [7] "Monoglobus.pectinilyticus" "Parasutterella.excrementihominis"
## [9] "Proteobacteria.bacterium.CAG.139" "Turicimonas.muris"
## [11] "X.Clostridium..leptum"
##
## $MEsalmon
## [1] "Bacteroides.faecis" "Bacteroides.faecis.CAG.32"
## [3] "Phascolarctobacterium.faecium"
##
## $MEtan
## [1] "Bacteroides.salyersiae" "Bifidobacterium.adolescentis"
## [3] "Bifidobacterium.bifidum" "Bifidobacterium.longum"
## [5] "Collinsella.aerofaciens"
##
## $MEturquoise
## [1] "Agathobaculum.butyriciproducens" "Anaerobutyricum.hallii"
## [3] "Anaerostipes.hadrus" "Bacteroides.eggerthii"
## [5] "Blautia.obeum" "Coprococcus.catus"
## [7] "Coprococcus.comes" "Dorea.formicigenerans"
## [9] "Dorea.longicatena" "Eubacterium.ramulus"
## [11] "Faecalibacterium.prausnitzii" "Fusicatenibacter.saccharivorans"
## [13] "Gemmiger.formicilis" "Roseburia.faecis"
## [15] "Roseburia.hominis" "Roseburia.inulinivorans"
## [17] "X.Eubacterium..rectale" "X.Ruminococcus..torques"
##
## $MEyellow
## [1] "Acidaminococcus.intestini" "Bacteroides.caccae"
## [3] "Bacteroides.ovatus" "Bacteroides.stercoris"
## [5] "Bacteroides.thetaiotaomicron" "Bacteroides.uniformis"
## [7] "Eubacterium.ventriosum" "Parabacteroides.distasonis"
## [9] "Parabacteroides.merdae" "Phocaeicola.vulgatus"
## [11] "Ruminococcaceae.bacterium.D16" "X.Ruminococcus..lactaris"We are now ready to run the MSEA analysis. We run \(100,000\) permutations to calculate the enrichment scores.
We can plot the enrichment scores to visualize the MSEA results.
p <- MSEA %>%
arrange(-pval) %>%
mutate(ID = factor(ID, levels = ID)) %>%
ggplot(aes(y = -log10(pval), x = ID)) +
geom_bar(stat = "identity", fill = 'cornflowerblue') + theme_bw() +
coord_flip() +
ggtitle('Statistically significant modules associated with disease') +
xlab('') +
ylab('MSEA enrichment score')
print(p)
Based on the MSEA results, we obtain 13 enriched modules of microbial species. We can also examine the members of the top enriched modules.
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"
## 2024-05-03 13:11:22.017144 INFO::Writing function arguments to log file
## 2024-05-03 13:11:22.020854 INFO::Verifying options selected are valid
## 2024-05-03 13:11:22.021348 INFO::Determining format of input files
## 2024-05-03 13:11:22.021789 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-05-03 13:11:22.027053 INFO::Formula for fixed effects: expr ~ disease + age + antibiotics_current_use
## 2024-05-03 13:11:22.027593 INFO::Factor detected for categorial metadata 'disease'. Provide a reference argument or manually set factor ordering to change reference level.
## 2024-05-03 13:11:22.028174 INFO::Filter data based on min abundance and min prevalence
## 2024-05-03 13:11:22.028605 INFO::Total samples in data: 136
## 2024-05-03 13:11:22.029042 INFO::Min samples required with min abundance for a feature not to be filtered: 13.600000
## 2024-05-03 13:11:22.034482 INFO::Total filtered features: 113
## 2024-05-03 13:11:22.035055 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
## 2024-05-03 13:11:22.041248 INFO::Total filtered features with variance filtering: 0
## 2024-05-03 13:11:22.04174 INFO::Filtered feature names from variance filtering:
## 2024-05-03 13:11:22.042158 INFO::Running selected normalization method: NONE
## 2024-05-03 13:11:22.042555 INFO::Applying z-score to standardize continuous metadata
## 2024-05-03 13:11:22.045912 INFO::Running selected transform method: LOG
## 2024-05-03 13:11:22.053563 INFO::Running selected analysis method: LM
## 2024-05-03 13:11:22.054091 INFO::Fitting model to feature number 1, PWY.1042..glycolysis.IV..plant.cytosol.
## 2024-05-03 13:11:22.056691 INFO::Fitting model to feature number 2, DTDPRHAMSYN.PWY..dTDP.L.rhamnose.biosynthesis.I
## 2024-05-03 13:11:22.058988 INFO::Fitting model to feature number 3, PWY.5695..urate.biosynthesis.inosine.5..phosphate.degradation
## 2024-05-03 13:11:22.061289 INFO::Fitting model to feature number 4, PWY.6936..seleno.amino.acid.biosynthesis
## 2024-05-03 13:11:22.063575 INFO::Fitting model to feature number 5, ILEUSYN.PWY..L.isoleucine.biosynthesis.I..from.threonine.
## 2024-05-03 13:11:22.065883 INFO::Fitting model to feature number 6, PWY.7111..pyruvate.fermentation.to.isobutanol..engineered.
## 2024-05-03 13:11:22.068172 INFO::Fitting model to feature number 7, VALSYN.PWY..L.valine.biosynthesis
## 2024-05-03 13:11:22.070453 INFO::Fitting model to feature number 8, PWY.6609..adenine.and.adenosine.salvage.III
## 2024-05-03 13:11:22.0727 INFO::Fitting model to feature number 9, PWY.6737..starch.degradation.V
## 2024-05-03 13:11:22.074986 INFO::Fitting model to feature number 10, PWY.7219..adenosine.ribonucleotides.de.novo.biosynthesis
## 2024-05-03 13:11:22.077257 INFO::Fitting model to feature number 11, PWY0.1586..peptidoglycan.maturation..meso.diaminopimelate.containing.
## 2024-05-03 13:11:22.079497 INFO::Fitting model to feature number 12, ASPASN.PWY..superpathway.of.L.aspartate.and.L.asparagine.biosynthesis
## 2024-05-03 13:11:22.081781 INFO::Fitting model to feature number 13, PWY.5973..cis.vaccenate.biosynthesis
## 2024-05-03 13:11:22.084004 INFO::Fitting model to feature number 14, PWY.7221..guanosine.ribonucleotides.de.novo.biosynthesis
## 2024-05-03 13:11:22.086273 INFO::Fitting model to feature number 15, PWY.5686..UMP.biosynthesis
## 2024-05-03 13:11:22.088552 INFO::Fitting model to feature number 16, X1CMET2.PWY..N10.formyl.tetrahydrofolate.biosynthesis
## 2024-05-03 13:11:22.090838 INFO::Fitting model to feature number 17, PWY.6122..5.aminoimidazole.ribonucleotide.biosynthesis.II
## 2024-05-03 13:11:22.093104 INFO::Fitting model to feature number 18, PWY.6277..superpathway.of.5.aminoimidazole.ribonucleotide.biosynthesis
## 2024-05-03 13:11:22.09538 INFO::Fitting model to feature number 19, PWY.6385..peptidoglycan.biosynthesis.III..mycobacteria.
## 2024-05-03 13:11:22.097716 INFO::Fitting model to feature number 20, PWY.3841..folate.transformations.II
## 2024-05-03 13:11:22.099989 INFO::Fitting model to feature number 21, RIBOSYN2.PWY..flavin.biosynthesis.I..bacteria.and.plants.
## 2024-05-03 13:11:22.102277 INFO::Fitting model to feature number 22, PWY.6387..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.I..meso.diaminopimelate.containing.
## 2024-05-03 13:11:22.104621 INFO::Fitting model to feature number 23, THISYNARA.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.III..eukaryotes.
## 2024-05-03 13:11:22.107479 INFO::Fitting model to feature number 24, PWY.7663..gondoate.biosynthesis..anaerobic.
## 2024-05-03 13:11:22.110268 INFO::Fitting model to feature number 25, PWY.6386..UDP.N.acetylmuramoyl.pentapeptide.biosynthesis.II..lysine.containing.
## 2024-05-03 13:11:22.112589 INFO::Fitting model to feature number 26, PWY.6700..queuosine.biosynthesis
## 2024-05-03 13:11:22.114873 INFO::Fitting model to feature number 27, PEPTIDOGLYCANSYN.PWY..peptidoglycan.biosynthesis.I..meso.diaminopimelate.containing.
## 2024-05-03 13:11:22.117176 INFO::Fitting model to feature number 28, TRNA.CHARGING.PWY..tRNA.charging
## 2024-05-03 13:11:22.119431 INFO::Fitting model to feature number 29, PWY.6121..5.aminoimidazole.ribonucleotide.biosynthesis.I
## 2024-05-03 13:11:22.121765 INFO::Fitting model to feature number 30, HISTSYN.PWY..L.histidine.biosynthesis
## 2024-05-03 13:11:22.124013 INFO::Fitting model to feature number 31, PWY.7229..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.I
## 2024-05-03 13:11:22.126292 INFO::Fitting model to feature number 32, PWY.7199..pyrimidine.deoxyribonucleosides.salvage
## 2024-05-03 13:11:22.128604 INFO::Fitting model to feature number 33, PANTO.PWY..phosphopantothenate.biosynthesis.I
## 2024-05-03 13:11:22.130898 INFO::Fitting model to feature number 34, PWY.2942..L.lysine.biosynthesis.III
## 2024-05-03 13:11:22.133182 INFO::Fitting model to feature number 35, PWY.7237..myo...chiro..and.scillo.inositol.degradation
## 2024-05-03 13:11:22.135418 INFO::Fitting model to feature number 36, PWY.6168..flavin.biosynthesis.III..fungi.
## 2024-05-03 13:11:22.137713 INFO::Fitting model to feature number 37, COA.PWY.1..coenzyme.A.biosynthesis.II..mammalian.
## 2024-05-03 13:11:22.139957 INFO::Fitting model to feature number 38, PWY.5667..CDP.diacylglycerol.biosynthesis.I
## 2024-05-03 13:11:22.142223 INFO::Fitting model to feature number 39, PWY0.1319..CDP.diacylglycerol.biosynthesis.II
## 2024-05-03 13:11:22.144458 INFO::Fitting model to feature number 40, PWY.5097..L.lysine.biosynthesis.VI
## 2024-05-03 13:11:22.146754 INFO::Fitting model to feature number 41, ANAGLYCOLYSIS.PWY..glycolysis.III..from.glucose.
## 2024-05-03 13:11:22.149032 INFO::Fitting model to feature number 42, PWY.6123..inosine.5..phosphate.biosynthesis.I
## 2024-05-03 13:11:22.151285 INFO::Fitting model to feature number 43, ARGININE.SYN4.PWY..L.ornithine.de.novo..biosynthesis
## 2024-05-03 13:11:22.153756 INFO::Fitting model to feature number 44, PWY.6163..chorismate.biosynthesis.from.3.dehydroquinate
## 2024-05-03 13:11:22.15602 INFO::Fitting model to feature number 45, THRESYN.PWY..superpathway.of.L.threonine.biosynthesis
## 2024-05-03 13:11:22.158304 INFO::Fitting model to feature number 46, PYRIDNUCSYN.PWY..NAD.biosynthesis.I..from.aspartate.
## 2024-05-03 13:11:22.160559 INFO::Fitting model to feature number 47, PWY.6124..inosine.5..phosphate.biosynthesis.II
## 2024-05-03 13:11:22.162851 INFO::Fitting model to feature number 48, PWY.6147..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.I
## 2024-05-03 13:11:22.165111 INFO::Fitting model to feature number 49, PWY.7539..6.hydroxymethyl.dihydropterin.diphosphate.biosynthesis.III..Chlamydia.
## 2024-05-03 13:11:22.167347 INFO::Fitting model to feature number 50, PWY.6151..S.adenosyl.L.methionine.cycle.I
## 2024-05-03 13:11:22.169636 INFO::Fitting model to feature number 51, SER.GLYSYN.PWY..superpathway.of.L.serine.and.glycine.biosynthesis.I
## 2024-05-03 13:11:22.171866 INFO::Fitting model to feature number 52, PWY.6126..superpathway.of.adenosine.nucleotides.de.novo.biosynthesis.II
## 2024-05-03 13:11:22.174131 INFO::Fitting model to feature number 53, PANTOSYN.PWY..pantothenate.and.coenzyme.A.biosynthesis.I
## 2024-05-03 13:11:22.176378 INFO::Fitting model to feature number 54, PWY.7228..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.I
## 2024-05-03 13:11:22.178637 INFO::Fitting model to feature number 55, COA.PWY..coenzyme.A.biosynthesis.I
## 2024-05-03 13:11:22.180865 INFO::Fitting model to feature number 56, PWY.4242..pantothenate.and.coenzyme.A.biosynthesis.III
## 2024-05-03 13:11:22.183188 INFO::Fitting model to feature number 57, PWY.6703..preQ0.biosynthesis
## 2024-05-03 13:11:22.185469 INFO::Fitting model to feature number 58, THISYN.PWY..superpathway.of.thiamin.diphosphate.biosynthesis.I
## 2024-05-03 13:11:22.187708 INFO::Fitting model to feature number 59, HISDEG.PWY..L.histidine.degradation.I
## 2024-05-03 13:11:22.189971 INFO::Fitting model to feature number 60, PWY.5659..GDP.mannose.biosynthesis
## 2024-05-03 13:11:22.192231 INFO::Fitting model to feature number 61, PWY.5030..L.histidine.degradation.III
## 2024-05-03 13:11:22.194489 INFO::Fitting model to feature number 62, PWY.5484..glycolysis.II..from.fructose.6.phosphate.
## 2024-05-03 13:11:22.196727 INFO::Fitting model to feature number 63, PWY.3001..superpathway.of.L.isoleucine.biosynthesis.I
## 2024-05-03 13:11:22.199008 INFO::Fitting model to feature number 64, PWY.6125..superpathway.of.guanosine.nucleotides.de.novo.biosynthesis.II
## 2024-05-03 13:11:22.201274 INFO::Fitting model to feature number 65, TRPSYN.PWY..L.tryptophan.biosynthesis
## 2024-05-03 13:11:22.203509 INFO::Fitting model to feature number 66, GLYCOLYSIS..glycolysis.I..from.glucose.6.phosphate.
## 2024-05-03 13:11:22.205782 INFO::Fitting model to feature number 67, PWY.724..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.II
## 2024-05-03 13:11:22.20804 INFO::Fitting model to feature number 68, PWY.6897..thiamin.salvage.II
## 2024-05-03 13:11:22.210317 INFO::Fitting model to feature number 69, BRANCHED.CHAIN.AA.SYN.PWY..superpathway.of.branched.amino.acid.biosynthesis
## 2024-05-03 13:11:22.212549 INFO::Fitting model to feature number 70, CALVIN.PWY..Calvin.Benson.Bassham.cycle
## 2024-05-03 13:11:22.214812 INFO::Fitting model to feature number 71, PWY.841..superpathway.of.purine.nucleotides.de.novo.biosynthesis.I
## 2024-05-03 13:11:22.217068 INFO::Fitting model to feature number 72, COMPLETE.ARO.PWY..superpathway.of.aromatic.amino.acid.biosynthesis
## 2024-05-03 13:11:22.219313 INFO::Fitting model to feature number 73, PWY.7220..adenosine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2024-05-03 13:11:22.221574 INFO::Fitting model to feature number 74, PWY.7222..guanosine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2024-05-03 13:11:22.224215 INFO::Fitting model to feature number 75, DENOVOPURINE2.PWY..superpathway.of.purine.nucleotides.de.novo.biosynthesis.II
## 2024-05-03 13:11:22.226496 INFO::Fitting model to feature number 76, PWY.7357..thiamin.formation.from.pyrithiamine.and.oxythiamine..yeast.
## 2024-05-03 13:11:22.228732 INFO::Fitting model to feature number 77, PWY.5103..L.isoleucine.biosynthesis.III
## 2024-05-03 13:11:22.230986 INFO::Fitting model to feature number 78, ARO.PWY..chorismate.biosynthesis.I
## 2024-05-03 13:11:22.233237 INFO::Fitting model to feature number 79, PWY.7282..4.amino.2.methyl.5.phosphomethylpyrimidine.biosynthesis..yeast.
## 2024-05-03 13:11:22.235467 INFO::Fitting model to feature number 80, PWY0.845..superpathway.of.pyridoxal.5..phosphate.biosynthesis.and.salvage
## 2024-05-03 13:11:22.237723 INFO::Fitting model to feature number 81, ANAEROFRUCAT.PWY..homolactic.fermentation
## 2024-05-03 13:11:22.239969 INFO::Fitting model to feature number 82, PWY.6892..thiazole.biosynthesis.I..E..coli.
## 2024-05-03 13:11:22.24223 INFO::Fitting model to feature number 83, RHAMCAT.PWY..L.rhamnose.degradation.I
## 2024-05-03 13:11:22.244487 INFO::Fitting model to feature number 84, PYRIDOXSYN.PWY..pyridoxal.5..phosphate.biosynthesis.I
## 2024-05-03 13:11:22.246777 INFO::Fitting model to feature number 85, PWY.1269..CMP.3.deoxy.D.manno.octulosonate.biosynthesis.I
## 2024-05-03 13:11:22.249074 INFO::Fitting model to feature number 86, PHOSLIPSYN.PWY..superpathway.of.phospholipid.biosynthesis.I..bacteria.
## 2024-05-03 13:11:22.251351 INFO::Fitting model to feature number 87, CITRULBIO.PWY..L.citrulline.biosynthesis
## 2024-05-03 13:11:22.253643 INFO::Fitting model to feature number 88, NAGLIPASYN.PWY..lipid.IVA.biosynthesis
## 2024-05-03 13:11:22.255883 INFO::Fitting model to feature number 89, BIOTIN.BIOSYNTHESIS.PWY..biotin.biosynthesis.I
## 2024-05-03 13:11:22.258155 INFO::Fitting model to feature number 90, PWY.5154..L.arginine.biosynthesis.III..via.N.acetyl.L.citrulline.
## 2024-05-03 13:11:22.2604 INFO::Fitting model to feature number 91, FASYN.ELONG.PWY..fatty.acid.elongation....saturated
## 2024-05-03 13:11:22.262666 INFO::Fitting model to feature number 92, PENTOSE.P.PWY..pentose.phosphate.pathway
## 2024-05-03 13:11:22.264949 INFO::Fitting model to feature number 93, PWYG.321..mycolate.biosynthesis
## 2024-05-03 13:11:22.267214 INFO::Fitting model to feature number 94, PWY.6519..8.amino.7.oxononanoate.biosynthesis.I
## 2024-05-03 13:11:22.269474 INFO::Fitting model to feature number 95, PWY.7664..oleate.biosynthesis.IV..anaerobic.
## 2024-05-03 13:11:22.271697 INFO::Fitting model to feature number 96, PWY.4981..L.proline.biosynthesis.II..from.arginine.
## 2024-05-03 13:11:22.273956 INFO::Fitting model to feature number 97, GLYCOGENSYNTH.PWY..glycogen.biosynthesis.I..from.ADP.D.Glucose.
## 2024-05-03 13:11:22.276261 INFO::Fitting model to feature number 98, PWY.7388..octanoyl..acyl.carrier.protein..biosynthesis..mitochondria..yeast.
## 2024-05-03 13:11:22.278542 INFO::Fitting model to feature number 99, NONOXIPENT.PWY..pentose.phosphate.pathway..non.oxidative.branch.
## 2024-05-03 13:11:22.28079 INFO::Fitting model to feature number 100, COBALSYN.PWY..adenosylcobalamin.salvage.from.cobinamide.I
## 2024-05-03 13:11:22.283038 INFO::Fitting model to feature number 101, PWY.5989..stearate.biosynthesis.II..bacteria.and.plants.
## 2024-05-03 13:11:22.28532 INFO::Fitting model to feature number 102, PWY.6282..palmitoleate.biosynthesis.I..from..5Z..dodec.5.enoate.
## 2024-05-03 13:11:22.287562 INFO::Fitting model to feature number 103, PWY0.862...5Z..dodec.5.enoate.biosynthesis
## 2024-05-03 13:11:22.289858 INFO::Fitting model to feature number 104, PWY.7400..L.arginine.biosynthesis.IV..archaebacteria.
## 2024-05-03 13:11:22.292095 INFO::Fitting model to feature number 105, ARGSYN.PWY..L.arginine.biosynthesis.I..via.L.ornithine.
## 2024-05-03 13:11:22.294379 INFO::Fitting model to feature number 106, PWY.4984..urea.cycle
## 2024-05-03 13:11:22.296623 INFO::Fitting model to feature number 107, FASYN.INITIAL.PWY..superpathway.of.fatty.acid.biosynthesis.initiation..E..coli.
## 2024-05-03 13:11:22.298915 INFO::Fitting model to feature number 108, PWY4FS.7..phosphatidylglycerol.biosynthesis.I..plastidic.
## 2024-05-03 13:11:22.30119 INFO::Fitting model to feature number 109, PWY4FS.8..phosphatidylglycerol.biosynthesis.II..non.plastidic.
## 2024-05-03 13:11:22.30347 INFO::Fitting model to feature number 110, ARGSYNBSUB.PWY..L.arginine.biosynthesis.II..acetyl.cycle.
## 2024-05-03 13:11:22.305736 INFO::Fitting model to feature number 111, PWY.6317..galactose.degradation.I..Leloir.pathway.
## 2024-05-03 13:11:22.308047 INFO::Fitting model to feature number 112, PWY66.422..D.galactose.degradation.V..Leloir.pathway.
## 2024-05-03 13:11:22.310348 INFO::Fitting model to feature number 113, GLUTORN.PWY..L.ornithine.biosynthesis
## 2024-05-03 13:11:22.312581 INFO::Fitting model to feature number 114, PWY.2941..L.lysine.biosynthesis.II
## 2024-05-03 13:11:22.31486 INFO::Fitting model to feature number 115, PWY0.1296..purine.ribonucleosides.degradation
## 2024-05-03 13:11:22.317184 INFO::Fitting model to feature number 116, NAD.BIOSYNTHESIS.II..NAD.salvage.pathway.II
## 2024-05-03 13:11:22.319447 INFO::Fitting model to feature number 117, HSERMETANA.PWY..L.methionine.biosynthesis.III
## 2024-05-03 13:11:22.321862 INFO::Fitting model to feature number 118, PWY0.162..superpathway.of.pyrimidine.ribonucleotides.de.novo.biosynthesis
## 2024-05-03 13:11:22.324116 INFO::Fitting model to feature number 119, PWY.7208..superpathway.of.pyrimidine.nucleobases.salvage
## 2024-05-03 13:11:22.326417 INFO::Fitting model to feature number 120, TEICHOICACID.PWY..teichoic.acid..poly.glycerol..biosynthesis
## 2024-05-03 13:11:22.328661 INFO::Fitting model to feature number 121, PWY.6305..putrescine.biosynthesis.IV
## 2024-05-03 13:11:22.331025 INFO::Fitting model to feature number 122, GLUCONEO.PWY..gluconeogenesis.I
## 2024-05-03 13:11:22.333318 INFO::Fitting model to feature number 123, PWY.621..sucrose.degradation.III..sucrose.invertase.
## 2024-05-03 13:11:22.335578 INFO::Fitting model to feature number 124, PWY.6901..superpathway.of.glucose.and.xylose.degradation
## 2024-05-03 13:11:22.337857 INFO::Fitting model to feature number 125, POLYISOPRENSYN.PWY..polyisoprenoid.biosynthesis..E..coli.
## 2024-05-03 13:11:22.340106 INFO::Fitting model to feature number 126, PWY.7323..superpathway.of.GDP.mannose.derived.O.antigen.building.blocks.biosynthesis
## 2024-05-03 13:11:22.342395 INFO::Fitting model to feature number 127, PWY.5941..glycogen.degradation.II..eukaryotic.
## 2024-05-03 13:11:22.344631 INFO::Fitting model to feature number 128, PWY.6527..stachyose.degradation
## 2024-05-03 13:11:22.346882 INFO::Fitting model to feature number 129, PWY0.1261..anhydromuropeptides.recycling
## 2024-05-03 13:11:22.349209 INFO::Fitting model to feature number 130, PWY66.400..glycolysis.VI..metazoan.
## 2024-05-03 13:11:22.351455 INFO::Fitting model to feature number 131, PWY0.166..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis..E..coli.
## 2024-05-03 13:11:22.353706 INFO::Fitting model to feature number 132, GLYCOLYSIS.E.D..superpathway.of.glycolysis.and.Entner.Doudoroff
## 2024-05-03 13:11:22.355947 INFO::Fitting model to feature number 133, PWY.7187..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.II
## 2024-05-03 13:11:22.358248 INFO::Fitting model to feature number 134, PWY.7456..mannan.degradation
## 2024-05-03 13:11:22.360539 INFO::Fitting model to feature number 135, PWY.7184..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.I
## 2024-05-03 13:11:22.362801 INFO::Fitting model to feature number 136, OANTIGEN.PWY..O.antigen.building.blocks.biosynthesis..E..coli.
## 2024-05-03 13:11:22.365093 INFO::Fitting model to feature number 137, COLANSYN.PWY..colanic.acid.building.blocks.biosynthesis
## 2024-05-03 13:11:22.367331 INFO::Fitting model to feature number 138, PWY.6545..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.III
## 2024-05-03 13:11:22.36963 INFO::Fitting model to feature number 139, PWY.6895..superpathway.of.thiamin.diphosphate.biosynthesis.II
## 2024-05-03 13:11:22.371894 INFO::Fitting model to feature number 140, PWY.6608..guanosine.nucleotides.degradation.III
## 2024-05-03 13:11:22.374202 INFO::Fitting model to feature number 141, TCA..TCA.cycle.I..prokaryotic.
## 2024-05-03 13:11:22.376504 INFO::Fitting model to feature number 142, PWY.6859..all.trans.farnesol.biosynthesis
## 2024-05-03 13:11:22.378812 INFO::Fitting model to feature number 143, PWY.5100..pyruvate.fermentation.to.acetate.and.lactate.II
## 2024-05-03 13:11:22.381089 INFO::Fitting model to feature number 144, PWY.6572..chondroitin.sulfate.degradation.I..bacterial.
## 2024-05-03 13:11:22.383379 INFO::Fitting model to feature number 145, PWY.6471..peptidoglycan.biosynthesis.IV..Enterococcus.faecium.
## 2024-05-03 13:11:22.385704 INFO::Fitting model to feature number 146, PWY.6470..peptidoglycan.biosynthesis.V...beta..lactam.resistance.
## 2024-05-03 13:11:22.388019 INFO::Fitting model to feature number 147, PWY.5188..tetrapyrrole.biosynthesis.I..from.glutamate.
## 2024-05-03 13:11:22.39033 INFO::Fitting model to feature number 148, GLUCUROCAT.PWY..superpathway.of..beta..D.glucuronide.and.D.glucuronate.degradation
## 2024-05-03 13:11:22.392573 INFO::Fitting model to feature number 149, PWY.6749..CMP.legionaminate.biosynthesis.I
## 2024-05-03 13:11:22.39489 INFO::Fitting model to feature number 150, P164.PWY..purine.nucleobases.degradation.I..anaerobic.
## 2024-05-03 13:11:22.397211 INFO::Fitting model to feature number 151, PWY.7198..pyrimidine.deoxyribonucleotides.de.novo.biosynthesis.IV
## 2024-05-03 13:11:22.399497 INFO::Fitting model to feature number 152, PWY.7211..superpathway.of.pyrimidine.deoxyribonucleotides.de.novo.biosynthesis
## 2024-05-03 13:11:22.401797 INFO::Fitting model to feature number 153, PWY66.409..superpathway.of.purine.nucleotide.salvage
## 2024-05-03 13:11:22.40408 INFO::Fitting model to feature number 154, GALACT.GLUCUROCAT.PWY..superpathway.of.hexuronide.and.hexuronate.degradation
## 2024-05-03 13:11:22.406371 INFO::Fitting model to feature number 155, UDPNAGSYN.PWY..UDP.N.acetyl.D.glucosamine.biosynthesis.I
## 2024-05-03 13:11:22.408658 INFO::Fitting model to feature number 156, PWY.6353..purine.nucleotides.degradation.II..aerobic.
## 2024-05-03 13:11:22.410937 INFO::Fitting model to feature number 157, PWY.7197..pyrimidine.deoxyribonucleotide.phosphorylation
## 2024-05-03 13:11:22.413272 INFO::Fitting model to feature number 158, PWY.7242..D.fructuronate.degradation
## 2024-05-03 13:11:22.415526 INFO::Fitting model to feature number 159, PWY.7196..superpathway.of.pyrimidine.ribonucleosides.salvage
## 2024-05-03 13:11:22.41781 INFO::Fitting model to feature number 160, PWY.5690..TCA.cycle.II..plants.and.fungi.
## 2024-05-03 13:11:22.420062 INFO::Fitting model to feature number 161, PWY.5177..glutaryl.CoA.degradation
## 2024-05-03 13:11:22.42237 INFO::Fitting model to feature number 162, PWY.6478..GDP.D.glycero..alpha..D.manno.heptose.biosynthesis
## 2024-05-03 13:11:22.424607 INFO::Fitting model to feature number 163, HEXITOLDEGSUPER.PWY..superpathway.of.hexitol.degradation..bacteria.
## 2024-05-03 13:11:22.426918 INFO::Fitting model to feature number 164, GALACTUROCAT.PWY..D.galacturonate.degradation.I
## 2024-05-03 13:11:22.429223 INFO::Fitting model to feature number 165, PWY.5104..L.isoleucine.biosynthesis.IV
## 2024-05-03 13:11:22.431479 INFO::Fitting model to feature number 166, PWY.5505..L.glutamate.and.L.glutamine.biosynthesis
## 2024-05-03 13:11:22.433758 INFO::Fitting model to feature number 167, P441.PWY..superpathway.of.N.acetylneuraminate.degradation
## 2024-05-03 13:11:22.43602 INFO::Fitting model to feature number 168, PWY.6606..guanosine.nucleotides.degradation.II
## 2024-05-03 13:11:22.438358 INFO::Fitting model to feature number 169, SALVADEHYPOX.PWY..adenosine.nucleotides.degradation.II
## 2024-05-03 13:11:22.440624 INFO::Fitting model to feature number 170, PWY.5005..biotin.biosynthesis.II
## 2024-05-03 13:11:22.442921 INFO::Fitting model to feature number 171, PWY.6549..L.glutamine.biosynthesis.III
## 2024-05-03 13:11:22.445207 INFO::Fitting model to feature number 172, GLCMANNANAUT.PWY..superpathway.of.N.acetylglucosamine..N.acetylmannosamine.and.N.acetylneuraminate.degradation
## 2024-05-03 13:11:22.447463 INFO::Fitting model to feature number 173, PWY0.1241..ADP.L.glycero..beta..D.manno.heptose.biosynthesis
## 2024-05-03 13:11:22.449765 INFO::Fitting model to feature number 174, PWY.6507..4.deoxy.L.threo.hex.4.enopyranuronate.degradation
## 2024-05-03 13:11:22.452026 INFO::Fitting model to feature number 175, GALACTARDEG.PWY..D.galactarate.degradation.I
## 2024-05-03 13:11:22.454498 INFO::Fitting model to feature number 176, GLUCARGALACTSUPER.PWY..superpathway.of.D.glucarate.and.D.galactarate.degradation
## 2024-05-03 13:11:22.456849 INFO::Fitting model to feature number 177, PWY.5676..acetyl.CoA.fermentation.to.butanoate.II
## 2024-05-03 13:11:22.459175 INFO::Fitting model to feature number 178, PWY.5347..superpathway.of.L.methionine.biosynthesis..transsulfuration.
## 2024-05-03 13:11:22.461496 INFO::Fitting model to feature number 179, PWY.5138..unsaturated..even.numbered.fatty.acid..beta..oxidation
## 2024-05-03 13:11:22.46383 INFO::Fitting model to feature number 180, MET.SAM.PWY..superpathway.of.S.adenosyl.L.methionine.biosynthesis
## 2024-05-03 13:11:22.46616 INFO::Fitting model to feature number 181, PWY66.399..gluconeogenesis.III
## 2024-05-03 13:11:22.468462 INFO::Fitting model to feature number 182, PWY0.1297..superpathway.of.purine.deoxyribonucleosides.degradation
## 2024-05-03 13:11:22.470765 INFO::Fitting model to feature number 183, METSYN.PWY..L.homoserine.and.L.methionine.biosynthesis
## 2024-05-03 13:11:22.4731 INFO::Fitting model to feature number 184, PWY.7332..superpathway.of.UDP.N.acetylglucosamine.derived.O.antigen.building.blocks.biosynthesis
## 2024-05-03 13:11:22.475388 INFO::Fitting model to feature number 185, PWY.6588..pyruvate.fermentation.to.acetone
## 2024-05-03 13:11:22.477712 INFO::Fitting model to feature number 186, PWY.6891..thiazole.biosynthesis.II..Bacillus.
## 2024-05-03 13:11:22.48001 INFO::Fitting model to feature number 187, GLUCARDEG.PWY..D.glucarate.degradation.I
## 2024-05-03 13:11:22.482382 INFO::Fitting model to feature number 188, METH.ACETATE.PWY..methanogenesis.from.acetate
## 2024-05-03 13:11:22.484663 INFO::Fitting model to feature number 189, PWY.7013..L.1.2.propanediol.degradation
## 2024-05-03 13:11:22.486973 INFO::Fitting model to feature number 190, PWY.6590..superpathway.of.Clostridium.acetobutylicum.acidogenic.fermentation
## 2024-05-03 13:11:22.489347 INFO::Fitting model to feature number 191, FAO.PWY..fatty.acid..beta..oxidation.I
## 2024-05-03 13:11:22.491648 INFO::Fitting model to feature number 192, PWY.6318..L.phenylalanine.degradation.IV..mammalian..via.side.chain.
## 2024-05-03 13:11:22.493963 INFO::Fitting model to feature number 193, CENTFERM.PWY..pyruvate.fermentation.to.butanoate
## 2024-05-03 13:11:22.496242 INFO::Fitting model to feature number 194, GOLPDLCAT.PWY..superpathway.of.glycerol.degradation.to.1.3.propanediol
## 2024-05-03 13:11:22.498593 INFO::Fitting model to feature number 195, PWY.5136..fatty.acid..beta..oxidation.II..peroxisome.
## 2024-05-03 13:11:22.500988 INFO::Fitting model to feature number 196, PWY66.391..fatty.acid..beta..oxidation.VI..peroxisome.
## 2024-05-03 13:11:22.503283 INFO::Fitting model to feature number 197, HOMOSER.METSYN.PWY..L.methionine.biosynthesis.I
## 2024-05-03 13:11:22.505648 INFO::Fitting model to feature number 198, METHGLYUT.PWY..superpathway.of.methylglyoxal.degradation
## 2024-05-03 13:11:22.507952 INFO::Fitting model to feature number 199, PWY.7288..fatty.acid..beta..oxidation..peroxisome..yeast.
## 2024-05-03 13:11:22.510305 INFO::Fitting model to feature number 200, PWY.7383..anaerobic.energy.metabolism..invertebrates..cytosol.
## 2024-05-03 13:11:22.512617 INFO::Fitting model to feature number 201, P162.PWY..L.glutamate.degradation.V..via.hydroxyglutarate.
## 2024-05-03 13:11:22.514982 INFO::Fitting model to feature number 202, PWY.4041...gamma..glutamyl.cycle
## 2024-05-03 13:11:22.517317 INFO::Fitting model to feature number 203, P108.PWY..pyruvate.fermentation.to.propanoate.I
## 2024-05-03 13:11:22.519678 INFO::Fitting model to feature number 204, PWY.5022..4.aminobutanoate.degradation.V
## 2024-05-03 13:11:22.522106 INFO::Fitting model to feature number 205, GLUDEG.I.PWY..GABA.shunt
## 2024-05-03 13:11:22.524466 INFO::Fitting model to feature number 206, PWY.5677..succinate.fermentation.to.butanoate
## 2024-05-03 13:11:22.52681 INFO::Fitting model to feature number 207, P163.PWY..L.lysine.fermentation.to.acetate.and.butanoate
## 2024-05-03 13:11:22.529164 INFO::Fitting model to feature number 208, PWY.3781..aerobic.respiration.I..cytochrome.c.
## 2024-05-03 13:11:22.531507 INFO::Fitting model to feature number 209, PWY.5083..NAD.NADH.phosphorylation.and.dephosphorylation
## 2024-05-03 13:11:22.533883 INFO::Fitting model to feature number 210, DAPLYSINESYN.PWY..L.lysine.biosynthesis.I
## 2024-05-03 13:11:22.536167 INFO::Fitting model to feature number 211, P461.PWY..hexitol.fermentation.to.lactate..formate..ethanol.and.acetate
## 2024-05-03 13:11:22.538543 INFO::Fitting model to feature number 212, PWY.1861..formaldehyde.assimilation.II..RuMP.Cycle.
## 2024-05-03 13:11:22.540858 INFO::Fitting model to feature number 213, PWY.7312..dTDP.D..beta..fucofuranose.biosynthesis
## 2024-05-03 13:11:22.543197 INFO::Fitting model to feature number 214, PWY.6630..superpathway.of.L.tyrosine.biosynthesis
## 2024-05-03 13:11:22.545607 INFO::Fitting model to feature number 215, PWY.6595..superpathway.of.guanosine.nucleotides.degradation..plants.
## 2024-05-03 13:11:22.547916 INFO::Fitting model to feature number 216, ARG.POLYAMINE.SYN..superpathway.of.arginine.and.polyamine.biosynthesis
## 2024-05-03 13:11:22.550268 INFO::Fitting model to feature number 217, PWY.7003..glycerol.degradation.to.butanol
## 2024-05-03 13:11:22.552659 INFO::Fitting model to feature number 218, P185.PWY..formaldehyde.assimilation.III..dihydroxyacetone.cycle.
## 2024-05-03 13:11:22.555015 INFO::Fitting model to feature number 219, POLYAMSYN.PWY..superpathway.of.polyamine.biosynthesis.I
## 2024-05-03 13:11:22.557337 INFO::Fitting model to feature number 220, PWY0.1298..superpathway.of.pyrimidine.deoxyribonucleosides.degradation
## 2024-05-03 13:11:22.559642 INFO::Fitting model to feature number 221, LACTOSECAT.PWY..lactose.and.galactose.degradation.I
## 2024-05-03 13:11:22.561968 INFO::Fitting model to feature number 222, PWY4LZ.257..superpathway.of.fermentation..Chlamydomonas.reinhardtii.
## 2024-05-03 13:11:22.564258 INFO::Fitting model to feature number 223, P161.PWY..acetylene.degradation
## 2024-05-03 13:11:22.566593 INFO::Fitting model to feature number 224, RUMP.PWY..formaldehyde.oxidation.I
## 2024-05-03 13:11:22.56888 INFO::Fitting model to feature number 225, PPGPPMET.PWY..ppGpp.biosynthesis
## 2024-05-03 13:11:22.57119 INFO::Fitting model to feature number 226, PWY.5304..superpathway.of.sulfur.oxidation..Acidianus.ambivalens.
## 2024-05-03 13:11:22.5735 INFO::Fitting model to feature number 227, PWY.6531..mannitol.cycle
## 2024-05-03 13:11:22.575779 INFO::Fitting model to feature number 228, FUC.RHAMCAT.PWY..superpathway.of.fucose.and.rhamnose.degradation
## 2024-05-03 13:11:22.5781 INFO::Fitting model to feature number 229, PWY.7315..dTDP.N.acetylthomosamine.biosynthesis
## 2024-05-03 13:11:22.580366 INFO::Fitting model to feature number 230, FUCCAT.PWY..fucose.degradation
## 2024-05-03 13:11:22.582678 INFO::Fitting model to feature number 231, PWY.7210..pyrimidine.deoxyribonucleotides.biosynthesis.from.CTP
## 2024-05-03 13:11:22.584971 INFO::Fitting model to feature number 232, FOLSYN.PWY..superpathway.of.tetrahydrofolate.biosynthesis.and.salvage
## 2024-05-03 13:11:22.587277 INFO::Fitting model to feature number 233, PWY.6612..superpathway.of.tetrahydrofolate.biosynthesis
## 2024-05-03 13:11:22.589624 INFO::Fitting model to feature number 234, PWY0.1061..superpathway.of.L.alanine.biosynthesis
## 2024-05-03 13:11:22.591896 INFO::Fitting model to feature number 235, PWY.5189..tetrapyrrole.biosynthesis.II..from.glycine.
## 2024-05-03 13:11:22.594177 INFO::Fitting model to feature number 236, PWY.6876..isopropanol.biosynthesis
## 2024-05-03 13:11:22.596505 INFO::Fitting model to feature number 237, PWY0.781..aspartate.superpathway
## 2024-05-03 13:11:22.598841 INFO::Fitting model to feature number 238, P4.PWY..superpathway.of.L.lysine..L.threonine.and.L.methionine.biosynthesis.I
## 2024-05-03 13:11:22.601137 INFO::Fitting model to feature number 239, PWY0.1479..tRNA.processing
## 2024-05-03 13:11:22.603471 INFO::Fitting model to feature number 240, GLYCOLYSIS.TCA.GLYOX.BYPASS..superpathway.of.glycolysis..pyruvate.dehydrogenase..TCA..and.glyoxylate.bypass
## 2024-05-03 13:11:22.605784 INFO::Fitting model to feature number 241, PYRIDNUCSAL.PWY..NAD.salvage.pathway.I
## 2024-05-03 13:11:22.608058 INFO::Fitting model to feature number 242, GLYCOCAT.PWY..glycogen.degradation.I..bacterial.
## 2024-05-03 13:11:22.610342 INFO::Fitting model to feature number 243, PWY.5723..Rubisco.shunt
## 2024-05-03 13:11:22.612623 INFO::Fitting model to feature number 244, TCA.GLYOX.BYPASS..superpathway.of.glyoxylate.bypass.and.TCA
## 2024-05-03 13:11:22.614934 INFO::Fitting model to feature number 245, PWY.7385..1.3.propanediol.biosynthesis..engineered.
## 2024-05-03 13:11:22.617371 INFO::Fitting model to feature number 246, PWY.7254..TCA.cycle.VII..acetate.producers.
## 2024-05-03 13:11:22.62017 INFO::Fitting model to feature number 247, GLYOXYLATE.BYPASS..glyoxylate.cycle
## 2024-05-03 13:11:22.623023 INFO::Fitting model to feature number 248, PWY.6731..starch.degradation.III
## 2024-05-03 13:11:22.625863 INFO::Fitting model to feature number 249, PWY.7328..superpathway.of.UDP.glucose.derived.O.antigen.building.blocks.biosynthesis
## 2024-05-03 13:11:22.628677 INFO::Fitting model to feature number 250, POLYAMINSYN3.PWY..superpathway.of.polyamine.biosynthesis.II
## 2024-05-03 13:11:22.631529 INFO::Fitting model to feature number 251, PWY.2723..trehalose.degradation.V
## 2024-05-03 13:11:22.634357 INFO::Fitting model to feature number 252, GLUCOSE1PMETAB.PWY..glucose.and.glucose.1.phosphate.degradation
## 2024-05-03 13:11:22.637185 INFO::Fitting model to feature number 253, PWY.5384..sucrose.degradation.IV..sucrose.phosphorylase.
## 2024-05-03 13:11:22.639984 INFO::Fitting model to feature number 254, PWY.7046..4.coumarate.degradation..anaerobic.
## 2024-05-03 13:11:22.642821 INFO::Fitting model to feature number 255, PWY66.389..phytol.degradation
## 2024-05-03 13:11:22.645639 INFO::Fitting model to feature number 256, PWY.5971..palmitate.biosynthesis.II..bacteria.and.plants.
## 2024-05-03 13:11:22.648457 INFO::Fitting model to feature number 257, PWY.6629..superpathway.of.L.tryptophan.biosynthesis
## 2024-05-03 13:11:22.651306 INFO::Fitting model to feature number 258, PWY.5345..superpathway.of.L.methionine.biosynthesis..by.sulfhydrylation.
## 2024-05-03 13:11:22.654155 INFO::Fitting model to feature number 259, SULFATE.CYS.PWY..superpathway.of.sulfate.assimilation.and.cysteine.biosynthesis
## 2024-05-03 13:11:22.656996 INFO::Fitting model to feature number 260, PWY.5838..superpathway.of.menaquinol.8.biosynthesis.I
## 2024-05-03 13:11:22.659813 INFO::Fitting model to feature number 261, PRPP.PWY..superpathway.of.histidine..purine..and.pyrimidine.biosynthesis
## 2024-05-03 13:11:22.662645 INFO::Fitting model to feature number 262, PWY.5845..superpathway.of.menaquinol.9.biosynthesis
## 2024-05-03 13:11:22.665507 INFO::Fitting model to feature number 263, PWY.6113..superpathway.of.mycolate.biosynthesis
## 2024-05-03 13:11:22.668332 INFO::Fitting model to feature number 264, PWY.5897..superpathway.of.menaquinol.11.biosynthesis
## 2024-05-03 13:11:22.671167 INFO::Fitting model to feature number 265, PWY.5898..superpathway.of.menaquinol.12.biosynthesis
## 2024-05-03 13:11:22.674017 INFO::Fitting model to feature number 266, PWY.5899..superpathway.of.menaquinol.13.biosynthesis
## 2024-05-03 13:11:22.676981 INFO::Fitting model to feature number 267, PWY.7117..C4.photosynthetic.carbon.assimilation.cycle..PEPCK.type
## 2024-05-03 13:11:22.679788 INFO::Fitting model to feature number 268, PWY.6969..TCA.cycle.V..2.oxoglutarate.ferredoxin.oxidoreductase.
## 2024-05-03 13:11:22.682628 INFO::Fitting model to feature number 269, PWY.6628..superpathway.of.L.phenylalanine.biosynthesis
## 2024-05-03 13:11:22.685472 INFO::Fitting model to feature number 270, PWY.5861..superpathway.of.demethylmenaquinol.8.biosynthesis
## 2024-05-03 13:11:22.68829 INFO::Fitting model to feature number 271, PWY.5862..superpathway.of.demethylmenaquinol.9.biosynthesis
## 2024-05-03 13:11:22.691145 INFO::Fitting model to feature number 272, FERMENTATION.PWY..mixed.acid.fermentation
## 2024-05-03 13:11:22.69399 INFO::Fitting model to feature number 273, PWY.241..C4.photosynthetic.carbon.assimilation.cycle..NADP.ME.type
## 2024-05-03 13:11:22.69681 INFO::Fitting model to feature number 274, SO4ASSIM.PWY..sulfate.reduction.I..assimilatory.
## 2024-05-03 13:11:22.699692 INFO::Fitting model to feature number 275, KETOGLUCONMET.PWY..ketogluconate.metabolism
## 2024-05-03 13:11:22.702531 INFO::Fitting model to feature number 276, PWY.5840..superpathway.of.menaquinol.7.biosynthesis
## 2024-05-03 13:11:22.705394 INFO::Fitting model to feature number 277, PWY.5850..superpathway.of.menaquinol.6.biosynthesis.I
## 2024-05-03 13:11:22.70823 INFO::Fitting model to feature number 278, PWY.5896..superpathway.of.menaquinol.10.biosynthesis
## 2024-05-03 13:11:22.711084 INFO::Fitting model to feature number 279, P105.PWY..TCA.cycle.IV..2.oxoglutarate.decarboxylase.
## 2024-05-03 13:11:22.713933 INFO::Fitting model to feature number 280, PWY.5913..TCA.cycle.VI..obligate.autotrophs.
## 2024-05-03 13:11:22.71678 INFO::Fitting model to feature number 281, PWY.561..superpathway.of.glyoxylate.cycle.and.fatty.acid.degradation
## 2024-05-03 13:11:22.719648 INFO::Fitting model to feature number 282, PWY.4702..phytate.degradation.I
## 2024-05-03 13:11:22.722605 INFO::Fitting model to feature number 283, PWY.6803..phosphatidylcholine.acyl.editing
## 2024-05-03 13:11:22.752825 INFO::Fitting model to feature number 284, PWY.7115..C4.photosynthetic.carbon.assimilation.cycle..NAD.ME.type
## 2024-05-03 13:11:22.755698 INFO::Fitting model to feature number 285, PWY.5918..superpathay.of.heme.biosynthesis.from.glutamate
## 2024-05-03 13:11:22.758039 INFO::Fitting model to feature number 286, PWY.7234..inosine.5..phosphate.biosynthesis.III
## 2024-05-03 13:11:22.760261 INFO::Fitting model to feature number 287, PWY.6285..superpathway.of.fatty.acids.biosynthesis..E..coli.
## 2024-05-03 13:11:22.762526 INFO::Fitting model to feature number 288, PWY.5860..superpathway.of.demethylmenaquinol.6.biosynthesis.I
## 2024-05-03 13:11:22.764777 INFO::Fitting model to feature number 289, PWY.5863..superpathway.of.phylloquinol.biosynthesis
## 2024-05-03 13:11:22.767044 INFO::Fitting model to feature number 290, HEME.BIOSYNTHESIS.II..heme.biosynthesis.I..aerobic.
## 2024-05-03 13:11:22.769327 INFO::Fitting model to feature number 291, PWY.821..superpathway.of.sulfur.amino.acid.biosynthesis..Saccharomyces.cerevisiae.
## 2024-05-03 13:11:22.771564 INFO::Fitting model to feature number 292, PWY.5675..nitrate.reduction.V..assimilatory.
## 2024-05-03 13:11:22.77383 INFO::Fitting model to feature number 293, PWY.5791..1.4.dihydroxy.2.naphthoate.biosynthesis.II..plants.
## 2024-05-03 13:11:22.77613 INFO::Fitting model to feature number 294, PWY.5837..1.4.dihydroxy.2.naphthoate.biosynthesis.I
## 2024-05-03 13:11:22.778401 INFO::Fitting model to feature number 295, PWY.5920..superpathway.of.heme.biosynthesis.from.glycine
## 2024-05-03 13:11:22.780662 INFO::Fitting model to feature number 296, PWY.7204..pyridoxal.5..phosphate.salvage.II..plants.
## 2024-05-03 13:11:22.782953 INFO::Fitting model to feature number 297, ENTBACSYN.PWY..enterobactin.biosynthesis
## 2024-05-03 13:11:22.785256 INFO::Fitting model to feature number 298, PWY0.1415..superpathway.of.heme.biosynthesis.from.uroporphyrinogen.III
## 2024-05-03 13:11:22.787514 INFO::Fitting model to feature number 299, HEMESYN2.PWY..heme.biosynthesis.II..anaerobic.
## 2024-05-03 13:11:22.78982 INFO::Fitting model to feature number 300, PWY.6284..superpathway.of.unsaturated.fatty.acids.biosynthesis..E..coli.
## 2024-05-03 13:11:22.792078 INFO::Fitting model to feature number 301, P42.PWY..incomplete.reductive.TCA.cycle
## 2024-05-03 13:11:22.794374 INFO::Fitting model to feature number 302, PWY.5656..mannosylglycerate.biosynthesis.I
## 2024-05-03 13:11:22.796623 INFO::Fitting model to feature number 303, PWY.5173..superpathway.of.acetyl.CoA.biosynthesis
## 2024-05-03 13:11:22.798919 INFO::Fitting model to feature number 304, ECASYN.PWY..enterobacterial.common.antigen.biosynthesis
## 2024-05-03 13:11:22.801216 INFO::Fitting model to feature number 305, AST.PWY..L.arginine.degradation.II..AST.pathway.
## 2024-05-03 13:11:22.803496 INFO::Fitting model to feature number 306, PWY3O.355..stearate.biosynthesis.III..fungi.
## 2024-05-03 13:11:22.805792 INFO::Fitting model to feature number 307, PWY0.42..2.methylcitrate.cycle.I
## 2024-05-03 13:11:22.808065 INFO::Fitting model to feature number 308, PWY.7094..fatty.acid.salvage
## 2024-05-03 13:11:22.810371 INFO::Fitting model to feature number 309, PWY.5747..2.methylcitrate.cycle.II
## 2024-05-03 13:11:22.812639 INFO::Fitting model to feature number 310, PWY.5367..petroselinate.biosynthesis
## 2024-05-03 13:11:22.814938 INFO::Fitting model to feature number 311, PWY0.1533..methylphosphonate.degradation.I
## 2024-05-03 13:11:22.817237 INFO::Fitting model to feature number 312, PWY.6823..molybdenum.cofactor.biosynthesis
## 2024-05-03 13:11:22.819513 INFO::Fitting model to feature number 313, PWY.5705..allantoin.degradation.to.glyoxylate.III
## 2024-05-03 13:11:22.821811 INFO::Fitting model to feature number 314, LPSSYN.PWY..superpathway.of.lipopolysaccharide.biosynthesis
## 2024-05-03 13:11:22.824074 INFO::Fitting model to feature number 315, REDCITCYC..TCA.cycle.VIII..helicobacter.
## 2024-05-03 13:11:22.826355 INFO::Fitting model to feature number 316, GLYCOL.GLYOXDEG.PWY..superpathway.of.glycol.metabolism.and.degradation
## 2024-05-03 13:11:22.828623 INFO::Fitting model to feature number 317, P122.PWY..heterolactic.fermentation
## 2024-05-03 13:11:22.830916 INFO::Fitting model to feature number 318, KDO.NAGLIPASYN.PWY..superpathway.of..Kdo.2.lipid.A.biosynthesis
## 2024-05-03 13:11:22.833216 INFO::Fitting model to feature number 319, PWY.7446..sulfoglycolysis
## 2024-05-03 13:11:22.835521 INFO::Fitting model to feature number 320, PWY490.3..nitrate.reduction.VI..assimilatory.
## 2024-05-03 13:11:22.837831 INFO::Fitting model to feature number 321, PWY.7209..superpathway.of.pyrimidine.ribonucleosides.degradation
## 2024-05-03 13:11:22.840105 INFO::Fitting model to feature number 322, UBISYN.PWY..superpathway.of.ubiquinol.8.biosynthesis..prokaryotic.
## 2024-05-03 13:11:22.842389 INFO::Fitting model to feature number 323, PWY0.41..allantoin.degradation.IV..anaerobic.
## 2024-05-03 13:11:22.844624 INFO::Fitting model to feature number 324, PWY.5855..ubiquinol.7.biosynthesis..prokaryotic.
## 2024-05-03 13:11:22.846997 INFO::Fitting model to feature number 325, PWY.5856..ubiquinol.9.biosynthesis..prokaryotic.
## 2024-05-03 13:11:22.849328 INFO::Fitting model to feature number 326, PWY.5857..ubiquinol.10.biosynthesis..prokaryotic.
## 2024-05-03 13:11:22.851592 INFO::Fitting model to feature number 327, PWY.6708..ubiquinol.8.biosynthesis..prokaryotic.
## 2024-05-03 13:11:22.853926 INFO::Fitting model to feature number 328, PWY.5692..allantoin.degradation.to.glyoxylate.II
## 2024-05-03 13:11:22.856283 INFO::Fitting model to feature number 329, URDEGR.PWY..superpathway.of.allantoin.degradation.in.plants
## 2024-05-03 13:11:22.858609 INFO::Fitting model to feature number 330, PWY0.1338..polymyxin.resistance
## 2024-05-03 13:11:22.860876 INFO::Fitting model to feature number 331, P221.PWY..octane.oxidation
## 2024-05-03 13:11:22.863188 INFO::Fitting model to feature number 332, THREOCAT.PWY..superpathway.of.L.threonine.metabolism
## 2024-05-03 13:11:22.865486 INFO::Fitting model to feature number 333, NONMEVIPP.PWY..methylerythritol.phosphate.pathway.I
## 2024-05-03 13:11:22.867772 INFO::Fitting model to feature number 334, PWY.7560..methylerythritol.phosphate.pathway.II
## 2024-05-03 13:11:22.870069 INFO::Fitting model to feature number 335, PWY.5121..superpathway.of.geranylgeranyl.diphosphate.biosynthesis.II..via.MEP.
## 2024-05-03 13:11:22.872345 INFO::Fitting model to feature number 336, PWY.7392..taxadiene.biosynthesis..engineered.
## 2024-05-03 13:11:22.87464 INFO::Fitting model to feature number 337, PWY.6143..CMP.pseudaminate.biosynthesis
## 2024-05-03 13:11:22.876912 INFO::Fitting model to feature number 338, PWY.6270..isoprene.biosynthesis.I
## 2024-05-03 13:11:22.879205 INFO::Fitting model to feature number 339, ALLANTOINDEG.PWY..superpathway.of.allantoin.degradation.in.yeast
## 2024-05-03 13:11:22.881483 INFO::Fitting model to feature number 340, PWY.6263..superpathway.of.menaquinol.8.biosynthesis.II
## 2024-05-03 13:11:22.883743 INFO::Fitting model to feature number 341, PWY.7371..1.4.dihydroxy.6.naphthoate.biosynthesis.II
## 2024-05-03 13:11:22.886027 INFO::Fitting model to feature number 342, ARGORNPROST.PWY..arginine..ornithine.and.proline.interconversion
## 2024-05-03 13:11:22.888291 INFO::Fitting model to feature number 343, PWY66.398..TCA.cycle.III..animals.
## 2024-05-03 13:11:22.890557 INFO::Fitting model to feature number 344, PWY.5464..superpathway.of.cytosolic.glycolysis..plants...pyruvate.dehydrogenase.and.TCA.cycle
## 2024-05-03 13:11:22.89281 INFO::Fitting model to feature number 345, ORNDEG.PWY..superpathway.of.ornithine.degradation
## 2024-05-03 13:11:22.895107 INFO::Fitting model to feature number 346, P23.PWY..reductive.TCA.cycle.I
## 2024-05-03 13:11:22.897399 INFO::Fitting model to feature number 347, PWY0.1277..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation
## 2024-05-03 13:11:22.899665 INFO::Fitting model to feature number 348, ARGDEG.PWY..superpathway.of.L.arginine..putrescine..and.4.aminobutanoate.degradation
## 2024-05-03 13:11:22.901973 INFO::Fitting model to feature number 349, ORNARGDEG.PWY..superpathway.of.L.arginine.and.L.ornithine.degradation
## 2024-05-03 13:11:22.9043 INFO::Fitting model to feature number 350, HCAMHPDEG.PWY..3.phenylpropanoate.and.3..3.hydroxyphenyl.propanoate.degradation.to.2.oxopent.4.enoate
## 2024-05-03 13:11:22.906577 INFO::Fitting model to feature number 351, PWY.6690..cinnamate.and.3.hydroxycinnamate.degradation.to.2.oxopent.4.enoate
## 2024-05-03 13:11:22.908839 INFO::Fitting model to feature number 352, PROTOCATECHUATE.ORTHO.CLEAVAGE.PWY..protocatechuate.degradation.II..ortho.cleavage.pathway.
## 2024-05-03 13:11:22.911136 INFO::Fitting model to feature number 353, P124.PWY..Bifidobacterium.shunt
## 2024-05-03 13:11:22.913412 INFO::Fitting model to feature number 354, PWY.622..starch.biosynthesis
## 2024-05-03 13:11:22.915656 INFO::Fitting model to feature number 355, PWY.7269..NAD.NADP.NADH.NADPH.mitochondrial.interconversion..yeast.
## 2024-05-03 13:11:22.917959 INFO::Fitting model to feature number 356, PWY.7316..dTDP.N.acetylviosamine.biosynthesis
## 2024-05-03 13:11:22.920221 INFO::Fitting model to feature number 357, PWY.7279..aerobic.respiration.II..cytochrome.c...yeast.
## 2024-05-03 13:11:22.922624 INFO::Fitting model to feature number 358, PWY.6837..fatty.acid.beta.oxidation.V..unsaturated..odd.number..di.isomerase.dependent.
## 2024-05-03 13:11:22.924875 INFO::Fitting model to feature number 359, X3.HYDROXYPHENYLACETATE.DEGRADATION.PWY..4.hydroxyphenylacetate.degradation
## 2024-05-03 13:11:22.927171 INFO::Fitting model to feature number 360, PWY.6953..dTDP.3.acetamido.3.6.dideoxy..alpha..D.galactose.biosynthesis
## 2024-05-03 13:11:22.929494 INFO::Fitting model to feature number 361, PWY.7616..methanol.oxidation.to.carbon.dioxide
## 2024-05-03 13:11:22.931764 INFO::Fitting model to feature number 362, PWY.5088..L.glutamate.degradation.VIII..to.propanoate.
## 2024-05-03 13:11:22.934064 INFO::Fitting model to feature number 363, PWY0.881..superpathway.of.fatty.acid.biosynthesis.I..E..coli.
## 2024-05-03 13:11:22.982195 INFO::Counting total values for each feature
## 2024-05-03 13:11:23.012713 INFO::Writing filtered data to file output_pwys/features/filtered_data.tsv
## 2024-05-03 13:11:23.051684 INFO::Writing filtered, normalized data to file output_pwys/features/filtered_data_norm.tsv
## 2024-05-03 13:11:23.090505 INFO::Writing filtered, normalized, transformed data to file output_pwys/features/filtered_data_norm_transformed.tsv
## 2024-05-03 13:11:23.14067 INFO::Writing residuals to file output_pwys/fits/residuals.rds
## 2024-05-03 13:11:23.164429 INFO::Writing fitted values to file output_pwys/fits/fitted.rds
## 2024-05-03 13:11:23.180837 INFO::Writing all results to file (ordered by increasing q-values): output_pwys/all_results.tsv
## 2024-05-03 13:11:23.187181 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
## 2024-05-03 13:11:23.187987 INFO::Writing heatmap of significant results to file: output_pwys/heatmap.pdf
## [1] "There are no associations to plot!"
## 2024-05-03 13:11:23.188946 INFO::Writing association plots (one for each significant association) to output folder: output_pwys
## 2024-05-03 13:11:23.192275 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-05-03 13:11:23.192821 INFO::Plotting data for metadata number 1, antibiotics_current_use
## 2024-05-03 13:11:23.193918 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"