# S Segment Nextclade Dataset - Implementation Notes

## Summary
For reassortment detection purposes, it is important to have the full S nextclade.

Successfully implemented S segment nextclade dataset for Lassa virus, inspired from the L segment build structure. The dataset includes 780 authentic sequences (same as ones live) downloaded from NCBI with natural length variation (2,388-3,571 bp).

## Key Achievements
- ✅ **All major lineages supported**: LI, LII, LIII, LIV_V, LVII
- ✅ **Proper lineage assignments** for majority of sequences
- ✅ **Fixed critical GFF3 formatting issue** (missing final newline)

## Files Created/Modified

### New S Segment Files:
- `nextclade/defaults/s/` - Complete configuration directory
- `nextclade_data/s/` - Published dataset directory
- Updated `nextclade/Snakefile` and `nextclade/defaults/config.yaml`

### Key Configuration:
- **Reference**: NC_004296 (Josiah S segment)
- **Default CDS**: NP (nucleoprotein)
- **Gene annotations**: NP (101-1810) and GPC (1872-3347, complement)

## Testing Results
Tested with 15 representative sequences across all lineages:

### ✅ **Working Correctly (12/15 sequences):**
- **Perfect lineage assignments** for LI, LII, LIII, LIV_V, LVII
- **Reasonable QC scores** (69-7744)
- **Expected mutation counts** for evolutionary distance

### ⚠️ **Issues Identified (3/15 sequences):**
Three sequences show anomalous behavior despite being in the training dataset:
- **MH887809** (LIII, 3243bp): Assigned to LIV_V, QC score 1,328,806
- **KF478765** (LIV_V, 3571bp): Correct assignment but QC score 1,445,800  
- **LT601602** (LVII, 3415bp): Assigned to LIV_V, QC score 1,430,448

### Investigation Notes:
- All problematic sequences are confirmed present in both `sequences.fasta` and `tree.json`
- Tree root sequence matches reference perfectly
- Issue appears sequence-specific rather than systematic
- Sequences have unusual lengths (shortest: 3243bp, longest: 3571bp vs reference: 3402bp)

## Critical Fix Applied
**GFF3 formatting**: Added missing final newline to `genome_annotation.gff3` file, which resolved parsing issues and improved overall performance.

## Next Steps
1. Monitor performance with larger sequence sets
2. Investigate specific issues with the 3 problematic sequences
3. Consider filtering extreme length variants if issues persist
4. Deploy to production when satisfied with performance

## Workflow Integration
The S segment is now fully integrated into the automated workflow and will be built alongside L and GPC segments in future runs.