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Pipeline parameters

Input/output options

FASTQ start (basecalled reads)

Option Description
--input Path to the samplesheet CSV (example: fastq.csv)
--outdir Output directory for results.

To autogenerate a samplesheet.csv from a folder of demultiplexed FASTQ files, run pip install . once at the repo root, then metatropics-samplesheet -i . from your FASTQ directory.

POD5 start (basecalling inside the pipeline)

Option Description
--input Path to the samplesheet CSV (example: POD5.csv).
--input_dir Input directory with POD5. Default: None.
--outdir Output directory for results. Use absolute paths on cloud storage.
--model Dorado model (fast, hac, or sup). Default: hac.
--kit_name Dorado --kit-name. Allowed: EXP-NBD103,EXP-NBD104,EXP-NBD114,EXP-NBD114-24,EXP-NBD196,EXP-PBC001,EXP-PBC096,SQK-16S024,SQK-16S114-24,SQK-LWB001,SQK-MLK111-96-XL,SQK-MLK114-96-XL,SQK-NBD111-24,SQK-NBD111-96,SQK-NBD114-24,SQK-NBD114-96,SQK-PBK004,SQK-PCB109,SQK-PCB110,SQK-PCB111-24,SQK-PCB114-24,SQK-RAB201,SQK-RAB204,SQK-RBK001,SQK-RBK004,SQK-RBK110-96,SQK-RBK111-24,SQK-RBK111-96,SQK-RBK114-24,SQK-RBK114-96,SQK-RLB001,SQK-RPB004,SQK-RPB114-24,TWIST-16-UDI,TWIST-96A-UDI,TWIST-96B-UDI,TWIST-96C-UDI,TWIST-96D-UDI,VSK-PTC001,VSK-VMK001,VSK-VMK004,VSK-VPS001. Default: TWIST-96A-UDI.

To autogenerate a POD5.csv template, run metatropics-samplesheet pod5 -i . from your POD5 directory. For TWIST UDI plates, run metatropics-samplesheet pod5 TWIST-96A-UDI to create run.txt, edit sample names and wells, then metatropics-samplesheet pod5 TWIST-96A-UDI run.txt to build POD5.csv.

fastq_pass start (on-device basecalled, demultiplex inside the pipeline)

Option Description
--input Path to the samplesheet CSV (example: POD5.csv).
--input_dir Path to the fastq_pass folder with basecalled reads (not yet demultiplexed).
--basecall Must be false to skip pipeline basecalling and run demultiplexing only.
--outdir Output directory for results.
--kit_name Dorado --kit-name (e.g. TWIST-96C-UDI). Pipeline container: Dorado 2.0. Default: TWIST-96A-UDI.

Use metatropics-samplesheet pod5 -i . from your fastq_pass folder to create a POD5.csv template there. For TWIST UDI plates, run metatropics-samplesheet pod5 TWIST-96A-UDI to create run.txt, edit sample names and wells, then metatropics-samplesheet pod5 TWIST-96A-UDI run.txt to build POD5.csv.

Basecalling on the instrument should keep barcodes intact (Dorado --no-trim); otherwise demultiplexing may classify most reads as unclassified.

Read processing options

Option Description
--minLength Min read length to analyse. Default: 200.
--quality Min base quality used by QC and variant filtering. Default: 15.
--front Bases to trim at 5′. Default: 25.
--tail Bases to trim at 3′. Default: 25.

Rarefaction options

Option Description
--rarefaction Rarefy each sample to a target number of bases. Default: true.
--target_bases Target bases per sample when rarefying (e.g. ~1M reads × 500 bp). Default: 5e8 bases.

Host depletion option

Option Description
--Human_host_fasta Optional FASTA for the human background removal step.
--Other_host_fasta Optional FASTA for an additional host background (e.g. mosquito, primate).
--Host Optional host keyword(s) to auto-download FASTA under Metatropics/Databases. Multiple hosts are supported and will be merged (e.g. human,aedes,culex). Supported keywords include human, pan, gorilla, orangutan, macaque, aedes, anopheles, culex, bat, rat, dog, cat, camel, goat, pig, cow, mouse, chicken.

Viral classifier options (Virasign)

Important options

Option Description
--virasign_database Database(s) (e.g. RVDB, RefSeq, or RVDB,RefSeq). Default: RVDB.
--virasign_db_dir Where Virasign databases are stored (defaults to Databases/).
--virasign_ultrasensitive Enable Virasign ultrasensitive mode. Default: false.
--virasign_blind Blind specific viral species from analysis (not in any output). Use Virasign abbreviations (e.g. HEP,HIV,HTLV,EBV,CMV,HPV) or full species names (comma-separated). To list abbreviations: virasign --blinding.

Advanced / less common options

Option Description
--virasign_rvdb_version RVDB release to use (optional).
--virasign_accessions Extra accessions to include (optional).
--virasign_min_identity Min alignment identity threshold (optional).
--virasign_min_mapped_reads Min mapped reads to report a hit (optional).
--virasign_coverage_depth Min per-position depth for coverage filtering (optional).
--virasign_coverage_breadth Min breadth (fraction) for coverage filtering (optional).
--virasign_min_nogr Min number of non-overlapping genomic regions (NoGR) required (optional).
--virasign_zscore Enable/disable Z-score background correction (optional; default: enabled / true). Set to false to disable.
--virasign_zscore_controls Override auto-detection with sample IDs and/or FASTQ paths (≥2 controls). Examples: --zscore-controls H20_1,H20_2,BG_1,BG_2, --zscore-controls /path/water1.fastq.gz,/path/water2.fastq.gz, or --zscore-controls water_controls.txt (one sample ID or path per line).
--virasign_threads Threads for Virasign (optional; defaults to the task CPUs).
--virasign_ram_gb Minimap2 RAM/GB hint (-I) for Virasign (optional).
--virasign_enable_clustering Enable clustering of references in database prep. Default: false.
--virasign_cluster_identity Clustering identity threshold (optional).
--virasign_max_ambiguous_fraction Max allowed ambiguous fraction when preparing DB (optional).

Variant calling options and consensus thresholds (Clair3)

Option Description
--clair3_model Optional override for the Nanopore model.
--clair3_min_mq Min mapping quality (MAPQ). Default: 15.
--clair3_min_bq Min base quality (BQ). Default: 15.
--clair3_min_alt_reads Min ALT-supporting reads. Default: 10.
--depth Min per-position depth. Default: 25.
--agreement Min VAF for applying variants into the consensus. Default: 0.7.