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End to End Pipeline

end2end executes the complete RolyPoly workflow from raw reads to virus identification.

Options

Common

  • -i, --input: Raw RNA-seq data (fastq/gz file or directory) (required)
  • -o, --output-dir: Output directory (default: current_directory_rp_e2e)
  • -t, --threads: Number of threads (default: 1)
  • -M, --memory: Memory allocation (default: "6g")
  • -D, --host: Host/contamination fasta file
  • --keep-tmp: Keep temporary files (flag)
  • --log-file: Path to log file (default: current_directory/rolypoly_pipeline.log)

Assembly

  • -A, --assembler: Assemblers to use (default: "spades,megahit,penguin")
  • -d, --post-processing: Post-processing mode for assembly outputs (e.g., none, rmdup, linclust)

Filtering

  • -Fm1, --filter1_nuc: First nucleic filter (default: "alnlen >= 120 & pident>=75")
  • -Fm2, --filter2_nuc: Second nucleic filter (default: "qcov >= 0.95 & pident>=95")
  • -Fd1, --filter1_aa: First amino acid filter (default: "length >= 80 & pident>=75")
  • -Fd2, --filter2_aa: Second amino acid filter (default: "qcovhsp >= 95 & pident>=80")
  • --dont-mask: Skip masking RNA virus-like sequences in host fasta (flag)
  • --mmseqs-args: Additional MMseqs2 arguments
  • --diamond-args: Additional Diamond arguments (default: "--id 50 --min-orf 50")
  • --db: Database to use (default: "all")
  • --skip-existing: Skip pipeline steps when expected output already exists

Pipeline Steps

  1. Read Filtering
  2. Quality control
  3. Host/DNA removal

  4. rRNA decontamination

  5. Assembly

  6. Multi-assembler processing
  7. Optional clustering

  8. Final assembly generation

  9. Assembly Filtering

  10. Nucleotide/protein filtering

  11. Host sequence removal

  12. Marker Gene Search

  13. RdRp identification

  14. Genome Annotation

  15. RNA structure prediction and annotation

  16. Protein domain and ORF prediction.

  17. Virus Characteristics

  18. Feature analysis
  19. Report generation

Output Structure

output_dir/
├── filtered_reads/
├── assembly/
│   └── final_assembly.fasta
├── marker_search_results/
│   └── <DB_NAME>_marker_search_results.tsv (raw marker search pyhmmer output).
├── viral_bins/ (note! binning is not yet implemented - each contig is reported as a separate bin for now.)
│   └── bins_summary.tsv
│   └── bin_<<###>>
│       └── bin_<<###>>.fasta
│       └── bin_<<###>>_characteristics.tsv
│       └── genome_annotation/
│           └── bin_<<###>>_genes.tsv \ .gff3 (user-specified format - only ORFs are reported)
│           └── bin_<<###>>_proteins.faa (amino acid sequences)
│           └── bin_<<###>>_genome_annotation.tsv \ .gff3 (user-specified format, RNA and protein annotations)
│           └── bin_<<###>>_ribozymes.tsv 
└── RolyPoly_end_to_end_report.tsv (pipeline summary, concatenated log files, version information, and used tools/dbs citations).

Citations

RolyPoly

  • RolyPoly: RNA virus identification pipeline
  • Citation: Neri, U., Bushnell, B., Roux, S., & Camargo, A. P., Steindorff, A.S., Coclet, C., (2024). RolyPoly: A pipeline for RNA virus identification from RNA-seq data. [Software]. Available from https://code.jgi.doe.gov/UNeri/rolypoly

Read Processing

  • BBMap: Read processing
  • Citation: https://sourceforge.net/projects/bbmap/files/BBMap_39.08.tar.gz
  • SeqKit: Sequence manipulation
  • Citation: https://doi.org/10.1002/imt2.191
  • NCBI datasets: Data retrieval
  • Citation: https://ftp.ncbi.nlm.nih.gov/pub/datasets/command-line/v2/linux-amd64/datasets

Assembly

  • SPAdes: Genome assembly
  • Citation: https://doi.org/10.1089/cmb.2012.0021
  • MEGAHIT: Metagenome assembly
  • Citation: https://doi.org/10.1093/bioinformatics/btv033
  • Penguin: Virus-aware assembly
  • Citation: https://doi.org/10.1101/2024.03.29.587318
  • Bowtie: Read mapping
  • Citation: https://doi.org/10.1186/gb-2009-10-3-r25

Marker Gene Search

  • pyhmmer: HMM searches
  • Citation: https://doi.org/10.1093/bioinformatics/btad214
  • pyrodigal: ORF prediction
  • Citation: https://doi.org/10.21105/joss.04296

Filtering

  • MMseqs2: Sequence searching
  • Citation: https://doi.org/10.1038/nbt.3988
  • DIAMOND: Protein searching
  • Citation: https://doi.org/10.1038/nmeth.3176
  • pyfastx: FASTA handling
  • Citation: https://doi.org/10.1093/bib/bbaa368

Databases

  • SILVA: rRNA decontamination
  • Citation: https://doi.org/10.1093/nar/gks1219
  • RefSeq: DNA filtering
  • Citation: https://doi.org/10.1093%2Fnar%2Fgkv1189
  • RdRp Databases:
  • NeoRdRp_v2.1: https://doi.org/10.1264/jsme2.ME22001
  • RdRp-scan: https://doi.org/10.1093/ve/veac082
  • RVMT: https://doi.org/10.1016/j.cell.2022.08.023
  • TSA_2018: https://doi.org/10.1093/molbev/msad060
  • Pfam_A_37: https://doi.org/10.1093/nar/gkaa913