With the organelle case validated, PhytoFlow moved to its first nuclear genome run. The goal was not a publishable genome, it was a pipeline validation: does the tool selection logic work, and does the output reflect real plant biology?
How PhytoFlow routed itself
One flag triggers the entire nuclear analysis mode. No manual tool selection. No config editing.
graph TD
A[Input: HiFi Reads] --> B[--genome_type nuclear]
B --> C[Hifiasm Assembly]
C --> D[Coverage + MapQ QC]
D --> F[Helixer gene prediction]
F --> G[eggNOG-mapper]
G --> H[527 proteins predicted\n62.6% annotated\nAGL18 detected]
style H fill:#2d5a27,color:#fff
The data
5,000 PacBio HiFi reads from Arabidopsis thaliana (ENA: ERR8666127), average length 18,236bp. A deliberately small dataset — the goal was pipeline validation, not a publishable genome.
Assembly results
Hifiasm produced 38 contigs totalling 2.1Mb. All 38 passed the Helixer minimum length filter (≥21kb) — the first biological checkpoint. Sequences too short do not have enough context for deep learning gene prediction.
What coverage reveals
Two contigs stood out: high coverage (73-162x) with low MapQ (~3). High coverage combined with low mapping quality is the signature of repetitive or organelle sequence. Plant HiFi reads always contain chloroplast and mitochondrial DNA — these contigs are likely organelle fragments assembled alongside nuclear sequence.
The remaining contigs showed 2-25x coverage with MapQ 45-60 — the expected signature of unique nuclear sequence at low read depth.
Gene prediction
Helixer predicted 527 proteins from 2.1Mb of sequence — approximately one gene per 4kb, consistent with the known Arabidopsis gene density of one gene per 4.5kb. The prediction density alone suggests these are real genes.
Functional annotation
eggNOG-mapper annotated 330 of 527 proteins (62.6%) with GO terms. The most represented pathway was MAP kinase signalling — Arabidopsis stress response genes. This makes biological sense for a partial assembly where stress-responsive loci are enriched.
Two genes stood out as validation signals:
AGL18 : a MADS-box transcription factor controlling floral development. AGL18 specifically acts as a floral repressor. Its detection in a 2.1Mb partial assembly from 5,000 reads is a strong validation signal — it is a well-characterised gene with a distinctive domain signature that Helixer and eggNOG both correctly identified.
TFIIB : transcription initiation factor B. Core transcription machinery, conserved across all eukaryotes. Its presence confirms the assembly contains genuinely nuclear sequence, not just organelle or repetitive DNA.
NLR-Annotator
Zero NLR loci detected. This is expected — NBS-LRR disease resistance genes are large, multi-domain proteins that require substantial nuclear contigs to be detected. With 2.1Mb of partial assembly the statistical power is not there. On a complete Arabidopsis genome you would expect approximately 150 NLR candidates.
What this validates
Case 2 confirms that the pipeline correctly:
- Auto-detects nuclear de novo mode from
--genome_type nuclear - Enables Helixer, disables MAKER automatically
- Skips scaffolding when no reference or Hi-C data is provided
- Predicts biologically plausible genes at the correct density
- Annotates known Arabidopsis genes with correct functions
The three cases so far
| Case 1 | Case 2 | |
|---|---|---|
| Data | Organelle HiFi | 5k nuclear HiFi |
| Extra input | None | None |
| Gene predictor | None | Helixer |
| Proteins predicted | 0 | 527 |
| Key finding | 100.8% chloroplast | AGL18 detected |
Next: Case 3 — nuclear + reference mode with BRAKER3.