grigsby.phd In-progress

ITS Processing Comparison Data

ITS Processing Comparison Data

This folder contains data from two ITS1 sequencing processing approaches:

Folders

Original data from first post/

Paired-end processing results (from 2026-02-23 post):

  • 397 total ASVs detected
  • Read retention: 50-75% (losses during merging step)
  • ITSxpress: Failed
  • Files:
    • feature-table.tsv - ASV × sample abundance matrix
    • genus_plot_data.csv - Taxonomy and abundance data
    • denoising-stats.csv - DADA2 processing statistics
    • alpha_diversity.csv - Alpha diversity metrics
    • genus_prevalence.csv - Genus prevalence data

Forward ONLY/

Forward-reads-only processing results (new analysis):

  • 1,640 total ASVs detected (4.1× increase)
  • Read retention: 90-96% (no merging step)
  • ITSxpress: Successful ITS1 extraction
  • Files:
    • feature-table.tsv - ASV × sample abundance matrix
    • taxonomy_fwd.tsv - QIIME2 taxonomy assignments
    • denoising_stats_fwd_run1.tsv - DADA2 stats for Run 1
    • denoising_stats_fwd_run2.tsv - DADA2 stats for Run 2
    • taxabarplot.csv - Taxonomy barplot data
    • Sequence summaries/ - Quality and length distribution data

Key Findings

Metric Paired-End Forward-Only Improvement
ASVs detected 397 1,640 4.1×
Mean read retention 58-62% 93-94% +35%
ITSxpress success No Yes ✓
Samples below threshold 8/58 (13.8%) 0/58 (0%) ✓

Visualizations Available

Current plots in /assets/js/2026-02-26-plots.js:

  1. ✅ Read retention comparison (scatter plot)
  2. ✅ ASV rank-abundance curves
  3. ✅ Phylum-level ASV counts (grouped bar chart)
  4. ✅ ITSxpress sequence length distribution
  5. ✅ Genus overlap Venn diagram

Data Processing

Paired-End Pipeline

  1. Import EMP paired-end sequences
  2. Demultiplex by barcodes
  3. Trim primers with Cutadapt
  4. DADA2 denoise-paired (includes merging step)
  5. Taxonomy assignment with UNITE
  6. Filter to Fungi only

Forward-Only Pipeline

  1. Import EMP single-end sequences (forward only)
  2. Demultiplex by barcodes
  3. Trim primers with Cutadapt
  4. ITSxpress ITS1 extraction (new step)
  5. DADA2 denoise-single (no merging)
  6. Taxonomy assignment with UNITE
  7. Filter to Fungi only

References