Shotgun Metagenomics
We provide complete analysis for shotgun metagenomic sequencing, from raw data to interactive visualizations. Our pipeline includes host removal, taxonomic classification, functional profiling, resistome/pathway analysis, and diversity assessment. This enables high-resolution insights into microbial composition and function.
Workflow Summary
--- config: theme: 'base' themeVariables: fontFamily: 'verdana' fontSize: '25px' --- flowchart LR subgraph Preprocessing["`**Preprocessing**`"] direction TB Raw["Raw Reads"] QC["Quality Control"] Trim["Adapter Trimming"] Host["Host Filtering"] Error["Error Correction (optional)"] Raw --> QC --> Trim --> Host --> Error end subgraph Taxonomy["`**Taxonomic Profiling**`"] direction TB Classify["Taxonomic Classification (e.g., Kraken2, MetaPhlAn)"] Agg["Abundance Table Aggregation"] Vis1["Composition Visualization"] Classify --> Agg --> Vis1 end subgraph Functional["`**Functional Profiling**`"] direction TB Annot["Functional Annotation (e.g., HUMAnN)"] Pathway["Pathway Inference (e.g., MetaCyc, KEGG)"] Resistome["Resistome/ARG Analysis (optional)"] Vis2["Functional Visualization"] Annot --> Pathway --> Resistome --> Vis2 end subgraph Diversity["`**Diversity Analysis**`"] direction TB Alpha["Alpha Diversity"] Beta["Beta Diversity"] Ord["Ordination (PCoA/NMDS)"] Vis3["Diversity Plots"] Alpha --> Beta --> Ord --> Vis3 end Preprocessing --> Taxonomy --> Functional --> Diversity
Preprocessing
- Raw Reads: Input raw FASTQ files from shotgun sequencing.
- Quality Control: Remove low-quality reads and sequencing artifacts.
- Adapter Trimming: Trim adapter sequences.
- Host Filtering: Remove host reads (e.g., human, mouse) using alignment tools like Bowtie2.
- Error Correction (optional): Improve read accuracy using tools like SPAdes corrector or BFC.
Taxonomic Profiling
- Taxonomic Classification: Classify microbial reads using Kraken2, Bracken, or MetaPhlAn.
- Abundance Table Aggregation: Summarize abundance by species, genus, or phylum.
- Visualization: Generate taxonomic barplots, Krona charts, and relative abundance heatmaps.
Functional Profiling
- Functional Annotation: Map reads to gene families using HUMAnN, eggNOG, or Diamond.
- Pathway Inference: Map genes to biological pathways (e.g., MetaCyc, KEGG).
- Resistome/ARG Analysis (optional): Identify antimicrobial resistance genes using tools like AMRPlusPlus or CARD.
- Functional Visualization: Create heatmaps, pathway abundance plots, and functional summaries.
Diversity Analysis
- Alpha Diversity: Assess species richness and evenness within samples.
- Beta Diversity: Compare community composition between groups.
- Ordination: Visualize dissimilarities using PCoA, NMDS, or t-SNE.
- Diversity Plots: Generate ordination plots and dendrograms for comparative analysis.
Example
Stacked bar plots reveal the relative abundance of microbial taxa at different ranks.
Functional heatmaps visualize the presence and abundance of microbial pathways or gene families.
PCoA plots depict sample-level differences in microbial composition based on Bray-Curtis or Jaccard distance.
For detailed options and customization, contact us.