Nextflow Pipeline for Oomycete Effector Analysis

Overview

This Nextflow pipeline performs a comprehensive, reproducible analysis of five oomycete proteomes to identify effectors, characterize their promoter architecture and examine their amino acid composition. It integrates multiple modules for secretion prediction, effector classification, ortholog identification, upstream sequence extraction, motif discovery, and expression integration. The workflow has been optimized to explore transcriptional regulation and sequence-level features associated with pathogenicity, extending to both classically secreted and non-secreted effector-like candidates. The nextflow pipeline (main.nf) contains the further information on step-by-step scripts usage within pipeline.

Workflow Summary

1. Download Proteomes

• Source: Ensembl Protists Release 59
• Using ftp for all genomes (For example: "https://ftp.ebi.ac.uk/ensemblgenomes/pub/release-60/protists/fasta/protists_stramenopiles1_collection/plasmopara_halstedii_gca_900000015/pep/Plasmopara_halstedii_gca_900000015.Plasmopara_halstedii_genome.pep.all.fa.gz"). Download five proteomes in data/proteomes
• Species:
  • Plasmopara halstedii (GCA_900000015.1)
  • Phytophthora sojae (GCA_000149755.1)
  • Phytophthora infestans (GCA_000142945.1)
  • Hyaloperonospora arabidopsidis (GCA_000173235.2)
  • Pythium ultimum (GCA_000143045.1)

2. Secretion Signal Prediction

• Tool: SignalP 6.0
• Script used:scripts/signalp_pipeline.py
• Output: secreted vs. non-secreted FASTA in directory results/

3. Transmembrane Domain Prediction

• Tool: DeepTM
• Script used:scripts/deeptm_pipeline.py
• Classification: SP+Glob, SP+TM, TM, Glob

4. Effector Prediction

• Tools:
  • POOE (via Kaggle notebook in repo: https://github.com/sakshianil/POOE_lite)
  • WideEffHunter v1.0 ( Script used: scripts/run_wideeffhunter.sh)
• Output: Effectors grouped as motif/domain/PHI hits/cysteine-rich

5. Ortholog Identification

• Source: Ensembl REST API
• Type: One-to-one orthologs only
• Script used: scripts/fetch_orthologs.sh
• Validation: MAFFT + Jalview + InterProScan domains

6. Upstream and Protein Sequence Retrieval

• Tool: Ensembl REST API
• Script used: scripts/fetch_upstream_protein.sh
• Region: 1000 bp upstream from TSS

7. Amino Acid Composition Analysis

• Script used : scripts/aminoacid_composition.R
• Results stored in results/aminoacid_composition/.
• Key insight: Amino acid profiles of signal peptide and conserved region, particularly hydrophobic and polar residue enrichment, correlate with promoter-level motif diversity and regulatory complexity across effector classes.

8. Motif Discovery and Annotation

• Tools: MEME Suite (MEME, FIMO, MAST, GOMO, AMA, TOMTOM)
• Script used: scripts/motif_analysis.sh
• Databases stored within resources folder (define path to all databases within script as well):
  • JASPAR 2024
  • ELM 2024
  • Custom GOMO dbs

9. Expression Data Integration

• Dataset: Pl. halstedii transcriptome (PRJEB49134)
• Tool: DESeq2 (pipeline adapted from https://github.com/sakshianil/Transcriptional_regulation_oomycetes)
• Use: Match conserved orthologs to time-series profiles

10. Visualization

• Tools:
  • R (ggplot2, pheatmap)
  • motifStack (for motif phylogeny and logos)
• Output: Clustered heatmaps and motif enrichment maps

Acknowledgments

• POOE: https://github.com/zzdlabzm/POOE
• Structural layout influenced by OpenAI support for reproducible pipelines.

GitHub Repository

https://github.com/sakshianil/POOE_lite https://github.com/sakshianil/Transcriptional_regulation_oomycetes

License

MIT License © 2025 Sakshi Bharti

How to Run

– The Nextflow main.nf script and config file