Modular Nextflow Pipeline for SRR → Assembly → Amplicons → Pairwise Matrix

This repository provides a modular, Singularity-based Nextflow (DSL2) pipeline for:

1. Downloading raw reads from NCBI SRA (SRR accessions)
2. Assembling reads with Shovill
3. Predicting in‑silico amplicons using EMBOSS primersearch
4. Generating a pairwise difference matrix from amplicon FASTA files

---

Pipeline stages and data flow

SRR list
   │
   ▼
fastqs/          (download)
   │
   ▼
assemblies/      (shovill)
   │
   ▼
amplicons/       (primersearch)
   │
   ▼
pairwise matrix  (CSV)

---

Directory structure

When all stages are run, outputs are written under a single --outdir.

results/
├── fastqs/
│   ├── SRR123_1.fastq.gz
│   ├── SRR123_2.fastq.gz
│   └── ...
├── assemblies/
│   │   └── SRR123_assembled.fasta
│   └── ...
├── primersearch/
│   ├── amplicon/
│   │   ├── SRR123_assembled_extractedAmplicons.fasta
│   │   ├── SRR123_assembled_not_matched_primers.txt
│   ├── SRR123_assembled.fasta
│   ├── SRR123.json
│   ├── SRR123.ps
│   └── ...
├── matrix_rows/
│   │   └── SRR123_assembled_extractedAmplicons.txt
│   └── ...
└── matrix/
    └── pairwise_diff_matrix.csv

---

Common usage patterns

1. Full pipeline: SRR → matrix

nextflow run main.nf \
   --srr_list <SRR list file (one SRR per row)> \
   --outdir $PWD/<output folder> (need to use absolute path, or prefix it with $PWD\
   --primers <primer file (3-column primersearch format)> \ 
    --output_csv pairwise_test_50srrs.csv 

2. Download-only (raw FASTQ files from SRA)

nextflow run main.nf -entry download_only \
    --srr_list srr_ids.txt \
    --outdir results

3. Assembly-only (skip SRA download)

nextflow run main.nf -entry assembly_only \
    --reads /path/to/fastqs \
    --outdir results

4. Download + assembly (da)

nextflow run main.nf -entry da \
    --srr_list srr_ids.txt \
    --outdir results

5. Primersearch-only

nextflow run main.nf -entry primersearch_only \
    --reads /path/to/assemblies \
    --primers primers.txt \
    --outdir results \

6. Pairwise-only

nextflow run main.nf -entry pairwise_only \
    --amplicon_folder $PWD/amplicons (need to use absolute path, or prefix it with $PWD \
    --primers primers.txt \
    --output_csv pairwise.csv \
    --outdir output_folder

7. Primersearch + pairwise matrix (pp)

nextflow run main.nf -entry pp \
    --reads assemblies \
    --primers primers.txt \
    --outdir $PWD/results  (need to use absolute path, or prefix it with $PWD\
    --output_csv pairwise.csv

---

Repository layout

.
├── main.nf
├── nextflow.config
├── modules/
│   ├── download_reads.nf
│   ├── assemble_shovill.nf
│   ├── primersearch_amplicons.nf
│   └── pairwise_matrix.nf
├── bin/
│   ├── parse_primersearch.py
│   ├── fasta_to_json.py
│   ├── pairwise_compare.py
│   └── merge_rows.py
└── README.md

---

Notices

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