Interplay between Non-Coding RNA Transcription, Stringent/Relaxed Phenotype and Antibiotic Production in Streptomyces ambofaciens.
The main result did not reproduce in this reproduction attempt. Where our recomputation produced values that differ from the published ones, those discrepancies are listed below. This is a single automated attempt — not peer review and not a finding of error or misconduct — and differences can also arise from data access, undocumented parameters or the computing environment. The verdict can be contested via “report an error”.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
PARTIAL 1:1-ish reproduction. The repo (epinatel/Bacterial_RNAseq) is a GENERIC bacterial RNA-seq protocol, not the paper's own code, so reproduced under P16 by running the paper's NAMED tools (Bowtie2 2.2.6, featureCounts subread 2.0.6, Rockhopper 2.0.3) on the assigned wild-type data PRJNA342588 (8 PAIRED runs, ~4 GB) vs genome GCF_001267885.1 on «our HPC» compute nodes. Structural pipeline outputs REPRODUCE: transcriptional-unit count ~5175 vs reported 5587 (within ~7%; 1339 vs 1154 polycistronic operons), and a comparable novel-ncRNA inventory (341 Rockhopper predicted RNAs = 178 intergenic + 163 antisense; antisense within ~20% of the reported 202 class I+II asRNAs). R3 QC is sane: Bowtie2 mean 66.2% overall alignment, ~96% properly paired, strand-specific featureCounts assigns mean 64.9% to genes, rRNA ~16-17%. NOT reproduced 1:1: the exact ncRNA class split (45 sRNA / 119 / 83 / 507 cutoRNA) and the 87->90.8% & 45->94% coverage figures, which depend on the authors' custom classification + annotation-merge steps not shipped in the generic repo; and the mutant DE (needs PRJNA430852, out of scope). No fabrication concern flagged — all reported values are plausibly derivable from this data with the named tools plus the authors' additional curation. Dataset PRJNA342588 profiled: 8 wt paired libraries complete, all QC checks pass, grade A.
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-25
- Rubric version
- not recorded
- Assessed by
- —
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether expression of non-coding RNAs (sRNAs and antisense RNAs) in Streptomyces ambofaciens is linked to the stringent/relaxed phenotype that controls antibiotic (spiramycin) production, potentially revealing novel effector mechanisms of the stringent response.
- ★ The S. ambofaciens ATCC 23877 transcriptome was redefined from RNAseq data into 5587 transcriptional units (4433 monocistronic, 1154 polycistronic) covering 90.8% of the linear chromosome method
- ★ 45 sRNAs and 119 antisense RNAs (asRNAs I) transcribed from dedicated promoters were identified, several unprecedented in Streptomyces finding
- ★ Predicted sRNA/asRNA targets encode proteins involved in transcription, translation, ribosomal structure/biogenesis, and morphological/biochemical differentiation finding
- ★ Expression of most ncRNAs was correlated with the stringent/relaxed phenotype across wild-type, ΔpirA (relaxed), and rpoB(R)-complemented (stringent) strains, suggesting novel effector mechanisms of the stringent response mechanism
- asRNAs III (cutoRNAs, from convergent gene overlap) are unusually abundant and uniformly distributed, a feature attributed to the high GC content and weak transcription termination typical of streptomycetes finding
- asRNA I-targeted CDSs are enriched in COG categories J (translation/ribosome biogenesis), F (nucleotide transport/metabolism), and C (energy production/conversion) relative to the whole genome finding
- About 70% of asRNAs I are evolutionarily conserved across Streptomyces and other genera, arguing against them being mere transcriptional noise finding
- scr5239, a previously characterized S. coelicolor sRNA homolog found in S. ambofaciens, is known to repress dagA (agarase) and metE (methionine synthase) mRNAs resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| bulk RNA-seq (transcriptome annotation/TU mapping) | Streptomyces ambofaciens ATCC 23877, grown in YS broth | none (growth time-course: 48h, 72h, 96h, 120h) | transcriptional unit structure, gene/UTR boundaries, ncRNA identification | Rockhopper software |
| RNA-seq differential expression | S. ambofaciens strains: wild type, ΩpirA mutant, ΩpirA + rpoB(R) derivative | pirA gene disruption (KO) and rpoB(R) mutant RNA polymerase gene introduction | differential expression of sRNAs and asRNAs relative to stringent/relaxed phenotype | — |
| in silico target prediction | S. ambofaciens predicted sRNAs and candidate target mRNAs | none (computational) | predicted sRNA-mRNA base-pairing interactions | IntaRNA software |
| phylogenetic conservation analysis | S. ambofaciens sRNA and asRNA I sequences vs. other Streptomyces/bacterial genomes | none (computational) | degree of sequence conservation across species/genera | GLASSgo software |
| functional/domain annotation search | S. ambofaciens predicted sRNAs and UTRs | none (computational) | identification of known ncRNA families, riboswitches, and RNA motifs | Rfam database |
- – 7229 originally annotated genes were restructured into 5587 transcriptional units (4433 monocistronic, 1154 polycistronic; max 23 genes/unit)
- ▲ Re-annotation increased the fraction of chromosome nucleotides included in the transcriptome 87% to 90.8% (3.8% increase)
- ▲ Reads mapping to annotated transcripts nearly doubled with the new annotation 45% to 94% on average
- – 162 new transcripts from dedicated promoters were classified as sRNAs and asRNAs I 45 sRNAs, 119 asRNAs I
- – A subset of sRNAs and asRNAs I showed clear differential expression across growth timepoints, mostly upregulated at later timepoints 11/49 (~25%) sRNAs and 39/119 (~33%) asRNAs I
- – IntaRNA identified putative mRNA targets for the detected sRNAs, including multiple sRNAs converging on single targets (e.g., Lrp, AcrR paralogs) 241 target mRNAs for 49 sRNAs
- – A substantial fraction of sRNAs were found to be highly conserved among Streptomyces species 26/49 sRNAs
- – Most asRNAs I were evolutionarily conserved rather than strain-specific 70% conserved (56.3% Streptomyces genus, 13.4% Streptomyces + other genera); 30% specific to S. ambofaciens
- count 7229 genes on the linear chromosome (original genome annotation)
- count 5587 transcriptional units (4433 monocistronic, 1154 polycistronic) (redefined transcriptome structure)
- other 53% (2944 genes) retained exactly original annotation (comparison of new vs. original gene annotation)
- other 87% to 90.8% of chromosome nucleotides included in transcriptome (effect of re-annotation on transcriptome coverage)
- other 45% to 94% average mapped reads falling into annotated transcripts (effect of re-annotation on read mapping)
- count 45 sRNAs and 119 asRNAs I identified from dedicated promoters (newly identified ncRNA classes)
- other 11/49 (~25%) sRNAs and 39/119 (~33%) asRNAs I differentially expressed during growth (growth-phase differential expression)
- count 241 putative target mRNAs predicted for 49 sRNAs (IntaRNA target prediction)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper used RNAseq to compare the transcriptome (including newly identified sRNAs and antisense RNAs) of three Streptomyces ambofaciens strains across four growth timepoints (48, 72, 96, 120 h), using Rockhopper software for transcript-unit annotation and differential-expression calls, GLASSgo for phylogenetic conservation analysis, and IntaRNA for sRNA-target prediction. Results were reported mainly as counts and percentages of transcripts meeting differential-expression or conservation criteria, and as COG category prevalence (Figure 4), without explicit p-values, dispersion measures, or effect sizes appearing in the provided text.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Rockhopper-based differential expression calling | sRNA and asRNA I expression across growth timepoints (48 vs 72/96/120 h), Figure 3 | — | not stated |
| COG functional enrichment (category prevalence comparison) | asRNA-targeted CDSs vs whole-genome CDSs, Figure 4 | — | not stated |
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Differential expression of ncRNAs across the four timepoints was determined via Rockhopper's built-in analysis and summarized as counts/percentages of transcripts showing 'clear differential expression.'↳ Could also: A dedicated RNA-seq differential-expression package such as DESeq2 or edgeR (e.g., Wald or likelihood-ratio test with Benjamini-Hochberg FDR correction) could also be applied to the same count data. — These tools report an explicit p-value/adjusted p-value and log2 fold-change per transcript, letting readers gauge both the statistical confidence and magnitude of each expression change alongside the reported percentages.
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COG functional enrichment of asRNA-targeted CDSs relative to whole-genome CDSs was described qualitatively as showing 'a prevalence' in certain categories.↳ Could also: A formal enrichment test such as Fisher's exact test or a hypergeometric test, optionally with FDR correction across the COG categories tested, could also be used. — This would provide a quantitative measure of whether the observed category prevalence exceeds chance expectation, with an associated p-value or adjusted p-value per category.
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Phylogenetic conservation of sRNAs/asRNAs was assessed with GLASSgo and summarized as percentages of sequences shared across taxonomic groups.↳ Could also: A bootstrap-supported phylogenetic conservation score or comparative genomics test could also be used. — This would attach a confidence measure to each conservation call rather than a single point estimate, which can help convey certainty for borderline cases.
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Strain/timepoint comparisons were reported without stating the number of biological or technical replicates in the provided text.↳ Could also: Explicitly reporting and analyzing a defined number of independent biological replicates (e.g., three or more per strain/timepoint) with a variance-aware statistical test could also be used. — This would enable dispersion estimates and confidence intervals around expression differences, supporting assessment of reproducibility across independent cultures.
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Proportions of differentially expressed or conserved transcripts were reported as simple counts and percentages (e.g., '11 out of 49 sRNAs').↳ Could also: Reporting a 95% confidence interval (e.g., exact binomial CI) around each proportion could also be used. — This would convey the precision of each estimated proportion, which is particularly informative when the underlying counts are small.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-34438997 (S. ambofaciens ncRNA / stringent-relaxed RNA-seq)
Paper: Pinatel et al., Antibiotics (Basel) 2021, 10.3390/antibiotics10080947. Code: github.com/epinatel/Bacterial_RNAseq (commit c8d8994, 2017) — a GENERIC bacterial RNA-seq protocol (1 file: RNAseq_analysis_pipeline.txt), NOT paper- specific scripts. Methods section names the concrete tools/params used. Data in OUR scope: SRA PRJNA342588 = WILD-TYPE ATCC 23877 only.
Data (PRJNA342588, wild-type)
8 biological samples = 4 timepoints (48/72/96/120 h) x 2 biol. replicates.
Each replicate deposited twice: a PAIRED run (SRR123986xx, canonical, matches
paper's featureCounts -p -B) and an older SINGLE copy (SRR423xxxx, identical
read_count). We use the 8 PAIRED runs.
48h : SRR12398690, SRR12398691
72h : SRR12398688, SRR12398689
96h : SRR12398686, SRR12398687
120h: SRR12398684, SRR12398685
Reference: GCF_001267885.1 / ASM126788v1 (ATCC 23877 linear chromosome).
Pipeline (from Methods)
trimming (implicit) -> Bowtie2 v2.2.6 -> featureCounts (Subread v2.0,
-F SAF -t gene -s 1 -T 15 -p -B -C -J --fracOverlap 0.5) -> DESeq2 v1.26.0;
ncRNA/TU: Rockhopper 2.0.3 per timepoint, merged + classified with BEDTools.
IN SCOPE (pipeline-derived, reproducible from wild-type data)
- R1 Transcriptome reconstruction / TUs (Rockhopper on wt timeseries): reported 5,587 TUs (4,433 monocistronic + 1,154 polycistronic); chromosome coverage 87% -> 90.8%. (Table 1)
- R2 ncRNA inventory (Rockhopper predicted RNAs + classification): 45 sRNAs; 119 class-I asRNAs; 83 class-II asRNAs; 507 class-III asRNAs.
- R3 Alignment / library QC (sanity): mapping rate, rRNA fraction, strand specificity, gene coverage — Bowtie2 + bedtools/featureCounts.
OUT OF SCOPE (cannot reproduce from PRJNA342588 alone)
- Differential expression sRNA/asRNA wt-vs-mutant (Suppl. Table S13): needs the MUTANT data PRJNA430852 (ΩpirA, ΩpirA rpoB(R)) — different BioProject, not the accession assigned to this room. [data_partial]
- IntaRNA sRNA target prediction (241 targets): downstream of sRNA set, external tool, sequence-based, not a quantitative pipeline output of the wt RNA-seq.
- All wet-lab results (HPLC antibiotic titres, growth curves, qRT-PCR). [wetlab]
Primary reproduction target
R1 + R2 via Rockhopper 2.0.3 on the 8 wt PAIRED runs vs GCF_001267885.1, with R3 alignment QC as supporting evidence.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
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