Transcriptome study · OPL_mRNA_3_24201
Diacamma assamense — Analysis steps
AZ1–AZ3 · Three individual whole-body workers from one colony · Mizoram University campus · Collected 1 October 2025 · Species: Diacamma assamense · Computational results; no experimental activity validation.
Follow the workflow from read processing and assembly through quality checks, annotation and antimicrobial peptide (AMP) screening. Each step links to the available methods or results.
| # | Step | Method / status | Result / notes | Files |
|---|---|---|---|---|
| Sequencing and assembly | ||||
| 01 | Read quality control | FastQC · TrimGalore | 73.42 million clean reads across AZ1–AZ3. Raw and trimmed FASTQ files were not delivered. | Original report Quality plot GC plot |
| 02 | De novo assembly | Trinity | 318,934 transcripts; 149,464,614 bases; 44.24% GC. The report’s unigene count uses an undocumented definition. | Transcript FASTA (in original ZIP) |
| 03 | Structural and functional annotation | TransDecoder · Trinotate | 41,281 predicted peptides. Functional annotation includes domain, GO and pathway references. | Peptide FASTA GFF3 Functional TSV |
| 04 | Read mapping and counts | HTSeq · original report | Reported mapping rates were 38.68% for AZ1, 32.37% for AZ2 and 38.92% for AZ3. The reference genome is unspecified and aligner descriptions conflict. Count tables use transcript isoform IDs. | AZ1 workbook AZ2 workbook AZ3 workbook |
| Quality checks and annotation | ||||
| 05 | Assembly and identifier audit | Completed · 4 Nov 2026 | 318,934 unique transcript IDs; no exact duplicate nucleotide sequences. N50 419 bp; median 375 bp. All 41,281 peptide IDs resolve to transcripts. N50 alone does not establish quality. | Audit results JSON Reproducible audit script |
| 06 | Expression-table integrity | Completed · retained counts | All IDs match the assembly; no duplicate IDs or invalid counts. AZ1: 27,922 rows; AZ2: 18,957; AZ3: 15,377. Union: 28,141 IDs; shared: 12,833. Union counts preserve NA for absent rows; no normalization or treatment comparison. | Count-table audit Delivered count union · TSV.gz |
| 07 | Assembly completeness assessment | BUSCO 6.1.0 (post-delivery) | 125 eukaryota_odb12.2 markers: 35.2% complete (28.8% single-copy, 6.4% duplicated), 30.4% fragmented, 34.4% missing. | Summary TXT Summary JSON Marker TSV Summary CSV Provenance Run script Environment Export script |
| 08 | Hymenoptera protein completeness | Completed · BUSCO 6.1.0 | 5,651 markers: 629 complete (11.1%), 404 fragmented (7.1%), 4,618 missing (81.7%). Protein-mode input and lineage differ from the earlier broad-eukaryote transcript run, so this is not a controlled before/after comparison. | BUSCO summary TXT BUSCO summary JSON All marker results Run provenance |
| 09 | Functional annotation and taxonomic review | DIAMOND · Swiss-Prot 2026_03 | Swiss-Prot matches covered 25,999 of 41,281 proteins and 51,062 of 318,934 transcripts. Bacterial homology flagged 12,398 protein records and 6,909 transcript records for review. Host and non-host assignment remains unresolved. | All protein annotations · TSV.gz All transcript annotations · TSV.gz Original AMP candidates Methods / reference checksums |
| 10 | Vector and adaptor screening | Completed · local UniVec review | 329 transcripts have moderate-or-strong score matches, including strong matches in 99. This is a local VecScreen-style implementation, not official FCS output. Biological homologues and library context require review before trimming. | Alignment review · TSV.gz Flagged transcript IDs Parameters and limitations |
| Antimicrobial peptide analysis | ||||
| 11 | Antimicrobial peptide screening | DRAMP matches | 688 match rows across 370 query peptides. Sequence matches are candidates for further validation. | AMP workbook |
| 12 | AMP sequence verification | Completed · candidate audit | All 370 query IDs match predicted peptides. 576/688 match rows have query fragments shorter than 20 aa (minimum 4 aa). Short matches require review; this cutoff is not a validated activity threshold. | AMP match audit 370 full candidate peptides |
| 13 | Integrated AMP candidate evidence | Completed · 4 Nov 2026 | 370 candidates linked to full peptides, ORF status and delivered counts. 143 complete ORFs; 123 with counts in all three samples. For 347 candidates all reported match fragments are under 20 aa. Flags guide review, not activity classification. Missing counts remain NA. | Candidate evidence TSV Join script |
| 14 | Full-sequence AMP alignment review | Completed · original target pairs | 688 full-candidate/DRAMP pairs re-aligned locally with BLOSUM62 (gap open −11, extend −0.5). Identity, query and target coverage are provided. No E-values; not a database-wide search or activity validation. | Alignment review TSV Methods and reference checksum Alignment script |
| 15 | Signal peptide and membrane topology | Completed · Phobius 1.01 | 370 candidates processed. Signal peptide predicted for 21; 18 have no predicted TM helix; 12 signal-positive candidates have complete ORFs. 64 candidates have a predicted TM helix. Missing N termini limit negative calls; predictions do not prove secretion or activity. | Candidate predictions TSV Original short predictions Raw model output Methods and limitations Prediction input FASTA Summary script |
| 16 | Historical secretion-based shortlist | Completed · evidence review | Historical tier: 11 complete and 7 partial ORFs with signal-peptide/no-TM predictions. Superseded by the broader homology and independent-model review below; this is not a validated AMP shortlist. | All 370 candidates 18 priority candidates Tiering script |
| 17 | Independent AMP prediction | Completed · ampir precursor model | All 41,281 proteins scored. At exploratory cutoff 0.8: 50 records (46 unique sequences) score above threshold; none of the original 370 does. Threshold sensitivity is supplied. Nine length-eligible records were also assessed by Macrel; long precursors were not forced into its mature-peptide model. | All scores · TSV.gz Original 370 scores Threshold sensitivity Scoped Macrel results Macrel eligibility Methods and limits |
| 18 | DRAMP database search | Completed · E-value-filtered search | 115 protein records matched the standardized DRAMP general reference at E ≤ 0.001, including 26 original candidates and one high ampir-score record. Short/modified reference exclusions limit detection; no-hit does not prove inactivity or novelty. | Database search alignments Search methods Reference exclusions |
| 19 | Integrated candidate review | Homology, ORF, topology and AMP scores | Three records with high scores, complete open reading frames, predicted signal peptides and zero predicted membrane helices encode the same peptide. Its best Swiss-Prot match was phage lambda spanin. The one high-score record with a DRAMP match had a partial ORF and a best Swiss-Prot match to human S100-A9. Ant origin and activity remain unverified. | Updated original 370 50 high-score records New-candidate Phobius output 50 predictor input sequences |
| Study records and manuscript | ||||
| 20 | Sample metadata and descriptive counts | Study records and expression workbooks | AZ1–AZ3 are three whole-body worker ants from one colony, collected at Mizoram University campus on 1 October 2025. The summaries describe observed counts and shared workbook entries. Missing entries are recorded as NA. | Sample metadata Count summaries Workbook overlap |
| 21 | Manuscript preparation | Draft and supporting materials | Includes the methods and results draft, figures, and a proposed validation study. Open items include access to the raw reads, clarification of source methods, host and non-host assignment, and experimental validation. | Methods and results draft Readiness and permitted claims Prospective validation design Homology figure Score sensitivity figure Figure captions Reproduction guide Research plan |
Original results ZIP · Original sequencing report · Checksums