Bio assembly qc

Bio Assembly QC

Assemble genomes/metagenomes and produce assembly QC artifacts.

Instructions

1. Validate the assembly manifest and inspect a restartable execution plan before starting expensive work:

   uv run --no-project python skills/bio-assembly-qc/scripts/run_assembly_qc.py \
     assemblies.tsv --out results/bio-assembly-qc
   uv run --no-project python skills/bio-assembly-qc/scripts/run_assembly_qc.py \
     assemblies.tsv --out results/bio-assembly-qc --execute
   

   The driver rejects samples whose upstream read_qc_status is not passed, normalizes assembler-specific outputs to per-sample contigs.fasta, chooses QUAST versus MetaQUAST from the declared mode, and reuses only non-empty declared outputs.

2. Select an assembler based on read type, genome/metagenome scope, and sample diversity:

   • Illumina short-read isolates and hybrid assemblies: SPAdes v4.0.0+ (final feature release; bug-fix-only series continues). Use metaSPAdes for short-read metagenomes.
   • Long-read bacterial isolates (PacBio CLR, ONT): Flye v2.9.5+ for the draft/baseline assembly. Use Autocycler v0.6+ when a complete, high-confidence bacterial consensus genome is needed from multiple independent long-read assembly attempts; do not use it for mixed-community metagenomes.
   • Long-read metagenomes: Flye v2.9.5+ in --meta mode (metaFlye) as the baseline for ONT/CLR mixed-community assemblies.
   • HiFi metagenomes: prefer metaMDBG v1.1 (~2× more circularized high-quality MAGs vs metaFlye on HiFi, better virus/plasmid recovery; Nature Biotechnology 2024, DOI: 10.1038/s41587-023-01983-6). Keep metaFlye as a comparator when a per-sample failure mode is suspected.
   • Diverse or very large long-read datasets where speed dominates: myloasm (2025) as a faster long-read metagenome assembler when its profile matches the dataset; document the choice in the run log.

3. Run assembly with resource-aware settings and record exact CLI, version, thread count, and RAM ceiling.

   • For very large ONT/metagenome FASTQs, use /bio-reads-qc-mapping guidance for filtering and avoid redundant full-file raw-read preflights before filtering. Record raw file metadata (stat path, size, mtime), optionally run a small sampled check, and write seqkit stats after each produced read set.
   • Use atomic output patterns for long-running filters and assemblies: write to .tmp, verify non-empty/readable output, then mv into the final path. Resume mode should skip existing final outputs only after sanity checks; when checks fail, use a tool-supported overwrite option or remove the corrupt final output before rerunning.
   • For Flye/metaFlye failures or interrupted jobs, prefer --resume or --resume-from in the existing output directory when the prior run is structurally intact. Do not delete a large partial assembly unless logs or missing stage files show it is corrupted.

4. Run QUAST v5.3+ (use MetaQUAST for metagenomes) and summarize metrics.

5. For every produced contigs.fasta, invoke /tracking-taxonomy-updates to run the BBTools-container QuickClade percontig domain screen before choosing downstream genome/MAG/viral/eukaryotic workflows.

6. Use the QuickClade domain routing table to decide the next step:

   • Bacteria/Archaea -> /bio-gene-calling, /bio-annotation, and GTDB-Tk taxonomy assignment.
   • Viral or virus-like -> /bio-viromics before prokaryotic MAG tooling.
   • Eukaryota -> eukaryote-aware gene/QC workflows and EukCC where bins or genomes are present.
   • Mixed/low-confidence -> split or flag contigs before domain-specific analysis.

Quick Reference

Task	Action
Run workflow	Follow the steps in this skill and capture outputs.
Validate inputs	Confirm required inputs and reference data exist.
Review outputs	Inspect reports and QC gates before proceeding.
Tool docs	See docs/README.md.

Input Requirements

Prerequisites:

• Tools declared in the project's pinned Pixi environment. See docs/README.md for expected tools.
• Sufficient disk and RAM for chosen assembler. Inputs:
• reads/.fastq.gz or reads/.fastq (raw or filtered reads; verify actual compression by content when suffixes are suspect).
• assemblies.tsv with sample_id, mode, read1, read2, and read_qc_status; supported core modes are short_isolate, long_isolate, short_metagenome, long_metagenome, and hifi_metagenome.

Output

• results/bio-assembly-qc/contigs.fasta
• results/bio-assembly-qc/assembly_metrics.tsv
• results/bio-assembly-qc/domain_routing.tsv
• results/bio-assembly-qc/qc_report.html
• results/bio-assembly-qc/logs/

Quality Gates

• Assembly size range and N50 distribution meet project thresholds.
• Every assembler output is normalized to a non-empty per-sample contigs.fasta before QC or downstream routing.
• On failure: retry with alternative parameters; if still failing, record in report and exit non-zero.
• Verify reads are present and readable. If gzip -t fails on a .gz-named file, inspect magic bytes or file type before labeling it corrupt; it may be plain FASTQ with the wrong suffix.
• Check available disk space before assembly.
• For large ONT/metagenome inputs, raw file metadata and post-filter seqkit stats are recorded without redundant full-file raw preflight scans.
• Long-running filter outputs use .tmp plus atomic rename, and resume guards distinguish valid completed outputs from partial/corrupt files.
• Flye/metaFlye logs are inspected before deciding whether to resume, rerun, or clean a partial output directory.
• For Autocycler isolate consensus, record each input assembler/run and confirm the sample is not a mixed community.
• QuickClade percontig domain screen completed or the reason for skipping it is explicitly recorded.
• Domain routing table is reviewed before selecting MAG, viral, bacterial/archaeal, or eukaryotic downstream tools.

Examples

Example 1: Expected input layout

reads/*.fastq.gz (raw reads).
assembler choice (spades | flye).

Use fixtures/assemblies.tsv as the executable short-read, long-read, and metagenome planning fixture.

Troubleshooting

Issue: Missing inputs or reference databases Solution: Verify paths and permissions before running the workflow.

Issue: Low-quality results or failed QC gates Solution: Review reports, adjust parameters, and re-run the affected step.

Issue: Large ONT assembly workflow appears stalled before assembly Solution: Check whether the script is doing a raw full-file preflight (gzip -t, raw seqkit stats) instead of productive filtering. For urgent routing/assembly, replace raw full scans with metadata plus sampled checks, then run filtering and post-filter stats.

Issue: Flye job timed out or was interrupted Solution: Inspect flye.log and stage files. If the output directory is intact, resubmit with Flye resume options rather than restarting from scratch.