This repository contains an analysis pipeline for 16S sequencing data developed for low biomass microbiome samples.
All code and methods were developed and are maintained by Jessica Whelan (jessica-whelan).
The pipeline is modular and under active development.
This repository contains the R code and analysis pipeline for 16S microbiome data analysis used for low biomass human samples. Below is a description of the main scripts and their intended purpose:
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16S-sequencing.R– The main analysis script for the project.- Performs preprocessing of raw 16S sequencing data, including quality filtering, decontamination, and normalization.
- Computes alpha and beta diversity metrics to assess microbial diversity within and between samples.
- Implements ML-based differential abundance analyses, allowing identification of taxa associated with experimental conditions.
- Serves as the central workflow for the project; additional functions and plots may be integrated as development progresses.
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IntraSampleVariability.R– Script to analyze for intra-sample variability when there are technical or biological replicates present- Filters low abundance taxa, agglomerates to desired taxa level
- Creates relative abundance barplots, ordination plots, calculates BC values and creates barplot + heatmap, and calculates alpha diversity values and barplot
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krona-plot-function.R– Interactive visualization module (in development).- Generates Krona HTML plots for hierarchical exploration of taxonomic abundance.
- Designed to provide an intuitive, interactive view of microbial composition for presentations or exploratory analysis.
- Once finalized, outputs will be incorporated into the main analysis script.
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testing-code.R– Development and testing script.- Contains code for various visualization options and exploratory analyses.
- Includes ANCOM-BC2 analysis for sensitivity testing of differential abundance results.
- Functions here are experimental and may be merged into the main workflow once validated.
Notes:
- All scripts are written in R and rely on packages such as
phyloseq,mixOmics, and other common microbiome analysis tools. Please see REFERENCES.md for references to R packages used in this pipeline that request citation
If you use this software in your research, please cite:
Cite all versions (concept DOI):
Whelan, J. (2025). Pipeline for 16S Sequencing Analysis of Human Microbiome Low Biomass Samples [Computer software].
https://doi.org/10.5281/zenodo.18771116
Version-specific
Whelan, J. (2026). Pipeline for 16S Sequencing Analysis of Human Microbiome Low Biomass Samples (Version 1.0.4) [Computer software].
https://doi.org/10.5281/zenodo.18771493
This pipeline was developed and is maintained by Jessica Whelan (jessica-whelan).
Any use, adaptation, redistribution, or incorporation of the code requires explicit written permission from the author and must include appropriate citation or acknowledgement.
Permission to use does not waive the requirement for appropriate citation or acknowledgement.
© 2025 Jessica Whelan. All rights reserved.