bio-workflows-edna-pipeline

name	bio-workflows-edna-pipeline
description	End-to-end eDNA metabarcoding from raw amplicons to community ecology. Covers QC, primer removal, denoising with OBITools3 or DADA2, contamination filtering, taxonomy assignment, Hill number diversity, and constrained ordination. Use when processing environmental DNA samples for biodiversity assessment or ecological surveys.
tool_type	mixed
primary_tool	obitools3
workflow	true
depends_on	["ecological-genomics/edna-metabarcoding","ecological-genomics/biodiversity-metrics","ecological-genomics/community-ecology","read-qc/quality-reports"]
qc_checkpoints	[{"after_demux":"Reads per sample >1000; negative controls <100 reads"},{"after_denoising":"Chimera rate <20%; ASV/OTU count reasonable for marker and environment"},{"after_decontam":"No unexpected taxa remaining in negative controls; tag-jumping artifacts removed"},{"after_taxonomy":"Assignment rate marker-specific: >90% to phylum for COI/BOLD, >60% for understudied markers"},{"after_diversity":"Rarefaction curves approaching asymptote; sample completeness >80%"}]

eDNA Metabarcoding Pipeline

Complete workflow from raw amplicon sequences to community ecology analysis, supporting both OBITools3 and DADA2 processing paths.

Pipeline Overview

Raw amplicon FASTQ (demultiplexed)
    |
    v
[1. QC] ------------------> FastQC / MultiQC quality assessment
    |
    v
[2. Primer Removal] ------> Cutadapt (remove forward + reverse primers)
    |                            |
    |                            +---> QC: reads per sample >1000
    |
    +--- Path A: OBITools3     +--- Path B: DADA2
    |       |                  |       |
    |       v                  |       v
    |   [3a. obi alignpairedend]   [3b. filterAndTrim]
    |       |                  |       |
    |       v                  |       v
    |   [4a. obi uniq]        |   [4b. learnErrors + dada]
    |       |                  |       |
    |       v                  |       v
    |   [5a. obi ecotag]      |   [5b. assignTaxonomy]
    |                          |
    +-------- Merge -----------+
                |
                v
[6. Contamination Filter] -> decontam / microDecon (negative control removal)
    |
    v
[7. Taxonomy Table] -------> Species x sample matrix
    |
    v
[8. Diversity Analysis] ---> iNEXT Hill numbers (q=0,1,2)
    |
    v
[9. Community Comparison] -> vegan CCA/RDA + indicspecies
    |
    v
Species table + diversity metrics + ordination plots

Step 1: Quality Assessment

fastqc -t 8 -o fastqc_output/ raw_reads/*.fastq.gz
multiqc fastqc_output/ -o multiqc_report/

Step 2: Primer Removal

# Adapter sequences are marker-specific; examples below for common eDNA markers
# --discard-untrimmed: remove reads without primers (likely off-target)
# --minimum-length 50: discard very short fragments after trimming

# COI (Leray primers mlCOIintF / jgHCO2198)
cutadapt -g GGWACWGGWTGAACWGTWTAYCCYCC -G TAIACYTCIGGRTGICCRAARAAYCA \
    --discard-untrimmed --minimum-length 50 -j 8 \
    -o trimmed/{sample}_R1.fastq.gz -p trimmed/{sample}_R2.fastq.gz \
    raw_reads/{sample}_R1.fastq.gz raw_reads/{sample}_R2.fastq.gz

Common primer sets by marker:

Marker	Forward Primer	Reverse Primer	Target
COI	mlCOIintF	jgHCO2198	Metazoan invertebrates
12S MiFish	MiFish-U-F	MiFish-U-R	Fish
ITS2	ITS3	ITS4	Fungi