16S/18S Amplicon Metagenomics

We offer high-quality 16S/18S rRNA amplicon sequencing analysis using a DADA2–phyloseq-based pipeline. From raw data to microbial profiling, functional prediction, and co-occurrence networks, our service enables comprehensive insights into microbial community structure and potential function.

Workflow Summary

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    themeVariables:
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flowchart LR
    subgraph Preprocessing["`**Preprocessing**`"]
        direction TB
        Raw["Raw Reads"]
        QC["Quality Filtering"]
        Trim["Primer/Adapter Removal"]
        Filter["Read Filtering & Truncation"]
        Denoise["ASV Inference"]
        Merge["Read Merging"]
        Chimera["Chimera Removal"]
        Taxa["Taxonomic Assignment"]
        Raw --> QC --> Trim --> Filter --> Denoise --> Merge --> Chimera --> Taxa
    end

    subgraph Phyloseq["`**Diversity**`"]
        direction TB
        Import["Import to phyloseq"]
        Agg["Taxonomic Aggregation"]
        Alpha["Alpha Diversity"]
        Beta["Beta Diversity"]
        Ord["Ordination (PCoA/NMDS)"]
        DA["Differential Abundance (DESeq2)"]
        Vis["Visualization"]
        Import --> Agg --> Alpha --> Vis
        Agg --> Beta --> Ord --> Vis
        Agg --> DA --> Vis
    end

    subgraph Functional["`**Function/network**`"]
        direction TB
        Picrust["Functional Prediction (PICRUSt2)"]
        FuncAgg["Pathway Aggregation"]        
        Net["Co-occurrence Network Construction"]
        Hub["Hub Taxa Detection"]
        CustomVis["Custom Visualization"]
        Picrust --> FuncAgg --> CustomVis
        Net --> Hub --> CustomVis
    end

    Preprocessing --> Phyloseq --> Functional

Preprocessing (DADA2)

1. Raw Reads: Input demultiplexed FASTQ files.
2. Quality Filtering: Visualize read quality and determine truncation thresholds.
3. Primer/Adapter Removal: Use cutadapt or dada2::removePrimers().
4. Read Filtering & Truncation: Based on quality profiles.
5. ASV Inference: Denoising and exact sequence variant calling using DADA2.
6. Read Merging: Merge paired-end reads.
7. Chimera Removal: Remove likely chimeric sequences.
8. Taxonomic Assignment: Assign taxonomy using SILVA, Greengenes, or PR2 databases.

Community Analysis (phyloseq)

1. Import to phyloseq: Create object linking ASV table, taxonomy, metadata.
2. Taxonomic Aggregation: Aggregate ASVs at genus, family, etc.
3. Alpha Diversity: Measure richness/evenness (Observed, Shannon, Simpson).
4. Beta Diversity: Distance-based comparisons (Bray-Curtis, Jaccard, UniFrac).
5. Ordination: Visualize community structure via PCoA, NMDS, PCA.
6. Differential Abundance: Detect significantly altered taxa (e.g., DESeq2).
7. Visualization: Generate publication-quality plots (barplots, heatmaps, ordination).

Functional & Network Analysis

1. Functional Prediction: Predict KEGG Orthologs and pathways using PICRUSt2 or Tax4Fun2.
2. Pathway Aggregation: Summarize functions by KO, pathway, or module level.
3. Functional Visualization: Create heatmaps, barplots, or clustering plots of pathways.
4. Co-occurrence Network: Infer microbial association networks using SparCC, SPIEC-EASI, or correlation-based methods.
5. Hub Taxa Detection: Identify central taxa using degree, betweenness, or eigenvector centrality.
6. Network Visualization: Visualize interactions using igraph, ggraph, or Cytoscape export.

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For detailed options and customization, contact us.