Introduction to microEDA • microEDA

microEDA simplifies exploratory data analysis (EDA) for microbiome datasets. Whether you’re working with MetaPhlAn outputs or phyloseq objects, microEDA provides a unified interface for visualization, basic quality control, and taxonomic filtering. This vignette demonstrates how to import data, create microEDA objects, and use core utility and plotting functions.

All functions work on both microEDA and phyloseq objects.

Import MetaPhlAn Profiles

Since the phyloseq package does not natively support MetaPhlAn profiles, microEDA provides custom functions to load these files and merge them into a single metaphlanProfile object.

A note on Database Version Compatibility: MetaPhlAn output files include a header line specifying the database version used for classification (e.g., #mpa_vJun23_CHOCOPhlAnSGB_202307). The load_metaphlan() function automatically extracts and stores this metadata.

When merging profiles with join_mpa_profiles(), the following rules apply:

Version Mismatch: Profiles with different database versions cannot be merged and will throw an error.
Invalid Inputs: Any input object that is not of class metaphlanProfile is ignored with a warning.

These checks ensure that only comparable profiles are merged, preventing errors arising from incompatible clade definitions or abundance normalizations.

library(microEDA)

# Load individual MetaPhlAn profiles
# load_metaphlan() automatically extracts the database version from the file header
mpa_merged <- load_metaphlan(system.file("extdata", "merged_abundance_table.txt", package = "microEDA"))

mpa_single <- load_metaphlan(system.file("extdata", "single_sample_profile.txt", package = "microEDA"))

# Merge profiles into a single metaphlanProfile object
# join_mpa_profiles() errors if database versions differ and warns on invalid inputs
mpa <- join_mpa_profiles(mpa_merged, mpa_single)

Create microEDA-class Objects

microEDA objects can be easily created for objects of class metaphlanProfile or phyloseq:

data(GlobalPatterns, package = "phyloseq")

me <- microEDA(GlobalPatterns)

mpa <- microEDA(mpa)

In turn, a microEDA or metaphlanProfile object can be converted to phyloseq with to_phyloseq():

ps <- to_phyloseq(me)

identical(GlobalPatterns, ps)
#> [1] TRUE

Utility Functions

QIIME-style two-underscore prefixes (e.g., “k__“,”p__“) can be…

# ...added to values in a taxonomyTable...
me <- add_taxonomy_prefix(me)
head(phyloseq::tax_table(me)[, c("Kingdom", "Phylum", "Order")])
#> Taxonomy Table:     [6 taxa by 3 taxonomic ranks]:
#>        Kingdom      Phylum             Order            
#> 549322 "k__Archaea" "p__Crenarchaeota" NA               
#> 522457 "k__Archaea" "p__Crenarchaeota" NA               
#> 951    "k__Archaea" "p__Crenarchaeota" "o__Sulfolobales"
#> 244423 "k__Archaea" "p__Crenarchaeota" NA               
#> 586076 "k__Archaea" "p__Crenarchaeota" NA               
#> 246140 "k__Archaea" "p__Crenarchaeota" NA


# ... and just as easily be removed.
me <- trim_taxonomy_prefix(me)
head(phyloseq::tax_table(me)[, c("Kingdom", "Phylum", "Order")])
#> Taxonomy Table:     [6 taxa by 3 taxonomic ranks]:
#>        Kingdom   Phylum          Order         
#> 549322 "Archaea" "Crenarchaeota" NA            
#> 522457 "Archaea" "Crenarchaeota" NA            
#> 951    "Archaea" "Crenarchaeota" "Sulfolobales"
#> 244423 "Archaea" "Crenarchaeota" NA            
#> 586076 "Archaea" "Crenarchaeota" NA            
#> 246140 "Archaea" "Crenarchaeota" NA

Inconsistencies (from, e.g., different database versions) in taxonomic annotations within the dataset at a specified rank can be identified with:

check_taxonomic_consistency(me, tax_rank = "Genus", detailed_report = TRUE)
#> Warning: Conflicting taxonomy for 8 taxa at rank 'Genus'. Inconsistent higher-level classifications detected for:
#> NA, Streptomyces, Clostridium, Mycoplana, Bacteroides, Eubacterium, Ruminococcus, Bacillus
#> Detailed differences (TRUE = inconsistent rank):
#> # A tibble: 8 × 6
#>   Genus   differs_in_Kingdom differs_in_Phylum differs_in_Class differs_in_Order
#>   <chr>   <lgl>              <lgl>             <lgl>            <lgl>           
#> 1 Bacill… FALSE              FALSE             FALSE            FALSE           
#> 2 Bacter… FALSE              TRUE              TRUE             TRUE            
#> 3 Clostr… FALSE              TRUE              TRUE             TRUE            
#> 4 Eubact… FALSE              FALSE             FALSE            FALSE           
#> 5 Mycopl… FALSE              FALSE             FALSE            TRUE            
#> 6 Rumino… FALSE              FALSE             FALSE            FALSE           
#> 7 Strept… FALSE              FALSE             FALSE            FALSE           
#> 8 NA      TRUE               TRUE              TRUE             TRUE            
#> # ℹ 1 more variable: differs_in_Family <lgl>

Taxa from a microEDA or phyloseq object can be filtered using minimum abundance and prevalence thresholds. Filtering can be applied globally or within groups defined by a metadata variable. For microEDA objects, filtered-out features can optionally be retained in the object’s info slot.

filtered_me <- filter_features(me, min_abundance = 100, min_prevalence = 0.1)
#> Total filtered features: 17734
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Filter parameters are recorded for microEDA objects and can be displayed directly with show_filter_history() or as part of a broader overview using summary():

summary(filtered_me)
#> Summary of microEDA object
#> ========================
#> Taxa:                1482
#> Samples:             26
#> Taxonomic Ranks:     7         Kingdom - Phylum - Class - Order - Family - Genus - Species
#> Sample Variables:    7         X.SampleID, Primer, Final_Barcode, Barcode_truncated_plus_T, Barcode_full_length, SampleType, Description
#> 
#> [Status: Raw Count Data]
#> Total Reads:         23,672,653
#> Min Count:           0
#> Max Count:           1,177,685
#> 
#> --- microEDA Info ---
#> taxrank:             Species
#> transforms:          <NULL>
#> filtered_taxa:       [ 17734 taxa by 7 taxonomic ranks in 26 samples ]
#> filter_history:      [ 1 filter step applied ] 
#> 
#>                      Parameter           :      Step 1
#>                      --------------------:-------------
#>                      Minimum abundance   :         100
#>                      Minimum prevalence  :         0.1
#>                      Filtered by group   :         N/A
#>                      Abundance criterion :  prevalence
#>                      Group requirement   :         N/A
#>                      Filtered kept       :        TRUE
#>                      Features removed    :       17734
#>                      Total removed       :       17734

The dataset can also be collapsed at specific ranks by aggregating features of the same taxonomy into higher-level taxonomic groups.

me_phylum <- agglomerate_taxa(me, tax_rank = "Phylum")
me_phylum
#> === microEDA - an extended phyloseq object ===
#> 
#> phyloseq-class experiment-level object
#> otu_table()   OTU Table:         [ 67 taxa and 26 samples ]
#> sample_data() Sample Data:       [ 26 samples by 7 sample variables ]
#> tax_table()   Taxonomy Table:    [ 67 taxa by 2 taxonomic ranks ]
#> phy_tree()    Phylogenetic Tree: [ 67 tips and 66 internal nodes ]
#> 
#> --- microEDA Info ---
#> taxrank: Phylum
#> transforms: <NULL>

Alternatively, the plot_taxa_... functions automatically invoke filter_features() and agglomerate_taxa() internally when relevant arguments (e.g., tax_rank) are provided, allowing to skip manual preprocessing.

Visualization Functions

plot_taxa_barchart(me, "p", as_relative = FALSE)
#> Warning in filter_features(me, min_abundance = min_abundance, min_prevalence =
#> min_prevalence, : Both 'min_abundance' and 'min_prevalence' are 0. No filtering
#> will be applied.
#> Taxa below 0.3% relative abundance were merged into 'Other'.
#> Plotting data...

me_subset <- phyloseq::subset_samples(me, SampleType %in% c("Soil", "Freshwater", "Ocean", "Mock"))

plot_taxa_heatmap(me_subset, "Species", group_var = "SampleType", prevalence_display = "fraction", rm_missing = TRUE, as_relative = TRUE)
#> Warning in filter_features(me, min_abundance = min_abundance, min_prevalence =
#> min_prevalence, : Both 'min_abundance' and 'min_prevalence' are 0. No filtering
#> will be applied.
#> Taxa below 6% relative abundance were merged into 'Other'.
#> Plotting data...

plot_taxa_sankey(GlobalPatterns, tax_rank = "Order", samples = "Even3", as_relative = TRUE)
#> Warning in filter_features(me, min_abundance = min_abundance, min_prevalence =
#> min_prevalence, : Both 'min_abundance' and 'min_prevalence' are 0. No filtering
#> will be applied.
#> Warning: Conflicting taxonomy for 1 taxon at rank 'Order'. Inconsistent higher-level classification detected for:
#> Unclassified_Actinobacteria
#> Taxa below 0.2% relative abundance were merged into 'Other'.

plot_taxa_upset(GlobalPatterns, "Phylum", "SampleType")
#> Warning in filter_features(me, min_abundance = min_abundance, min_prevalence =
#> min_prevalence, : Both 'min_abundance' and 'min_prevalence' are 0. No filtering
#> will be applied.
#> Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
#> ℹ Please use `linewidth` instead.
#> ℹ The deprecated feature was likely used in the ComplexUpset package.
#>   Please report the issue at
#>   <https://github.com/krassowski/complex-upset/issues>.
#> This warning is displayed once per session.
#> Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
#> generated.

To retrieve the specific names of taxa shared between or unique to groups, use get_taxa_overlaps() instead of plot_taxa_upset():

# Calculate overlaps for Phyla across SampleTypes
overlaps <- get_taxa_overlaps(GlobalPatterns, "Phylum", "SampleType",
  min_abundance = 100, min_prevalence = 3
)
#> Total filtered features: 17734
#> Filtered features: 549322, 522457, 951, 244423, 586076, 246140, 143239, 244960, 255340, 144887, 141782, 215972, 31759, 30678, 138353, 406058, 30405, 1126, 46043, 512519, 554594, 210741, 550229, 105798, 183473, 541313, 540305, 511243, 568829, 185612, 321018, 549041, 153762, 155789, 155495, 1029, 155526, 197473, 559394, 341551, 330416, 573586, 568724, 175045, 552540, 546313, 548602, 564501, 47778, 54107, 25116, 252853, 100679, 540349, 1140, 101071, 1143, 141739, 1141, 237125, 1144, 512943, 235345, 141417, 268471, 395774, 142115, 239672, 462844, 243675, 444679, 540290, 523571, 545095, 105679, 105066, 515962, 525569, 238793, 538819, 515691, 525464, 529521, 202343, 455926, 282132, 254458, 45742, 441638, 251993, 462098, 570593, 159421, 104332, 137608, 200619, 568129, 348720, 528115, 257655, 173903, 47016, 548723, 542636, 526502, 311512, 132088, 202347, 152431, 272691, 236606, 525, 52710, 592689, 55681, 206978, 467541, 566371, 453468, 557121, 524986, 102663, 264985, 37119, 349670, 240012, 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# View taxa specific to "Soil"
overlaps[["Soil-specific"]]
#> [1] "Elusimicrobia"

# View taxa shared by all groups
overlaps[["all-intersection"]]
#> [1] "Actinobacteria"  "Bacteroidetes"   "Cyanobacteria"   "Firmicutes"     
#> [5] "Proteobacteria"  "Verrucomicrobia"