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User guide

This section explains how to prepare, run, inspect, and interpret an MTD Explorer analysis.

Use this page as a map of the documentation.

Start here

Prepare an analysis

Set up input FASTQ files, samplesheets, metadata, and analysis modes.

Input files · Analysis modes

Run from the command line

Review the main options used to control host, microbiome, functional, and integration analyses.

Command-line reference

Understand the output folder

Learn how the major result folders are organized before opening the detailed output pages.

Output files

Check reproducibility

Inspect run parameters, software paths, database settings, and analysis metadata.

Methods and reproducibility outputs

Output guides

  • Taxonomic exploratory outputs

    Exploratory taxonomic summaries, read composition, abundance landscapes, diversity plots, overlap diagrams, and matrix QC.

    Open guide

  • Taxonomic visualizations

    Interactive Krona plots and GraPhlAn cladograms for inspecting taxonomic composition.

    Open guide

  • Host expression outputs

    Host count matrices, PCA, heatmaps, differential expression, volcano plots, and host functional enrichment.

    Open guide

  • Microbiome comparison outputs

    Group comparisons for non-host taxa, including heatmaps, alpha diversity, ANOSIM, ANCOM-BC, and microbiome differential abundance.

    Open guide

  • Functional profiling outputs

    Non-host functional profiles derived from HUMAnN, including GO and KEGG heatmaps and PCA plots.

    Open guide

  • ssGSEA outputs

    Sample-level host gene-set activity summaries, PCA, correlation heatmaps, variable gene-set heatmaps, and differential boxplots.

    Open guide

  • HAllA integration outputs

    Host–microbiome and host–function association summaries produced with HAllA.

    Open guide

  • Academic references

    Citation-ready references for the original MTD pipeline and the main software packages used by MTD Explorer.

    Open references

For a first complete analysis, read the documentation in this order:

1. Installation
2. Verify installation
3. Input files
4. Analysis modes
5. Command-line reference
6. Output files
7. Taxonomic exploratory outputs
8. Taxonomic visualizations
9. Host expression outputs
10. Microbiome comparison outputs
11. Functional profiling outputs
12. ssGSEA outputs
13. HAllA integration outputs
14. Methods and reproducibility outputs
15. Academic references

Result layers

MTD Explorer produces several result layers from the same RNA-seq dataset.

Result layer Main folder or file Main guide
Input setup samplesheet.txt, FASTQ files, metadata Input files
Analysis settings command-line options Command-line reference
Custom host setup Create_custom_host.sh, HostSpecies.csv Custom host references
Custom microbiome setup Create_custom_micro.sh, --kraken-micro-db, Kraken 2, Bracken Custom microbiome references
Supported hosts HostSpecies.csv Supported host species
Output structure main output directory Output files
Taxonomic exploration exploratory/taxonomy/ Taxonomic exploratory outputs
Taxonomic visualization krona/, graphlan/ Taxonomic visualizations
Host expression Host_DEG/ Host expression outputs
Microbiome comparison Nonhost_DEG/ Microbiome comparison outputs
Functional profiling hmn_genefamily_abundance_files/ Functional profiling outputs
Gene-set activity ssGSEA/ ssGSEA outputs
Multi-layer integration halla/ HAllA integration outputs
Reproducibility methods/ Methods and reproducibility outputs

Choosing the right page

Question Start with
How do I format my samplesheet? Input files
Should I run comparison or exploratory mode? Analysis modes
How do I create a host reference for my species? Custom host references
How do I create a custom Kraken 2/Bracken microbiome database? Custom microbiome references
Which host Taxon IDs are curated? Supported host species
How do I add a non-model host species? Custom host references
Which command-line option controls trimming, Kraken2, Bracken, or read layout? Command-line reference
Where are the main results saved? Output files
How do I inspect detected microbiome composition? Taxonomic exploratory outputs
Where are Krona and GraPhlAn plots? Taxonomic visualizations
Where are host DEG plots and tables? Host expression outputs
Where are ANOSIM and ANCOM-BC outputs? Microbiome comparison outputs
Where are GO/KEGG functional profiles? Functional profiling outputs
Where are host gene-set activity plots? ssGSEA outputs
Where are host–microbiome association outputs? HAllA integration outputs
Which tools should I cite? Academic references

Interpretation principles

MTD Explorer outputs are designed to be inspected together.

A taxonomic signal should be interpreted with read composition, abundance tables, database settings, and sample metadata.

A host expression signal should be interpreted with the count matrix, normalization, PCA, differential-expression tables, and biological context.

Functional and association outputs should be treated as exploratory summaries unless supported by the corresponding tables, statistical results, and study design.