Introduction

After eDNA is collected, it’s processed in the lab and analyzed through a series of molecular and computational steps. This section introduces the full pipeline from extraction to ecological insight.

DNA Extraction

Methods

  • Commercial kits (e.g., Qiagen, Zymo)
  • CTAB protocol (especially for difficult substrates)
  • Mechanical lysis (bead beating) for tough samples

Museum Considerations

  • Ethanol extracts may require extra clean-up
  • Small sample volumes and degraded DNA need gentle handling

PCR Amplification

Key Components

  • Target gene (COI, 16S, 18S, ITS)
  • Forward and reverse primers with sample-specific barcodes
  • Polymerase mix, buffers, thermal cycler

Multiplexing

  • Add indexes (barcodes) to each sample
  • Allows pooling of hundreds of samples for sequencing

Library Preparation & Sequencing

Platforms

  • Illumina MiSeq: Short reads, high accuracy, widely used
  • Oxford Nanopore: Long reads, portable, real-time
  • PacBio: Long, accurate reads (more expensive)

Library Steps

  • Clean and quantify PCR products
  • Pool amplicons
  • Add adapters for platform-specific sequencing

Bioinformatics Workflow

Quality Control

  • Trim adapters, remove low-quality reads
  • Filter out chimeras (artifacts)

Sequence Inference

  • Denoising tools (e.g., DADA2, UNOISE)
  • Generate ASV table (amplicon sequence variants)

Taxonomic Assignment

  • Compare ASVs to reference databases (SILVA, MIDORI, BOLD)
  • Use BLAST, RDP classifier, or QIIME’s built-in classifiers

Diversity and Community Analysis

Alpha Diversity

  • Measures richness within a sample
  • Shannon, Simpson, Observed ASVs

Beta Diversity

  • Compares composition between samples
  • Dissimilarity indices (Bray-Curtis, Jaccard)
  • Ordination techniques (NMDS, PCA, PCoA)

Visualization and Interpretation

Common Tools

  • phyloseq in R
  • Bar plots, heatmaps, diversity plots
  • Network analysis for co-occurrence

Museum Applications

  • Archive eDNA extracts with specimen metadata
  • Link modern and historical biodiversity
  • Contribute to public reference libraries (e.g., GenBank, BOLD)

Challenges and Caveats

  • False positives/negatives from contamination or low biomass
  • Transport effects: DNA from upstream or other locations
  • PCR bias: Not all taxa amplify equally

Preservation and DNA Recovery: Formalin and Ethanol

Why this matters:

  • Many museum collections contain valuable specimens preserved decades ago.
  • Modern methods like eDNA offer a chance to recover biodiversity signals from these specimens.
  • Success depends on how the material was stored and preserved.

DNA from Ethanol

  • DNA can leach into ethanol from tissue over time.
  • Ethanol can be extracted directly and filtered like a water sample.
  • Be aware of:
    • Evaporation
    • Microbial contamination
    • Loss of long DNA fragments

DNA from Formalin

  • Formalin causes extensive crosslinking, degrading DNA quality.
  • Extraction success depends on:
    • Sample age
    • Buffering (neutral-buffered formalin is better)
    • Use of reversal techniques (e.g., heat, alkaline lysis, long incubation)

Tips for Success

  • Use high-sensitivity kits or protocols for low-input DNA
  • Consider using PCR-free library prep if DNA is heavily damaged
  • Focus on short amplicons (100–200 bp) for degraded samples

Foundational Reading

Final Thoughts

eDNA analysis is a powerful tool, but it requires rigorous protocols and thoughtful interpretation. Used well, it offers scalable, reproducible insights across fields—from conservation to curation.