Read summary

Read quality and alignment statistics generated by fastcat bamstats for each input sample. Shown are distributions of read length and quality score, and a summary of alignment outcomes (mapped, unmapped, primary, supplementary) against the reference genome.

ref sample_name total primary secondary supplementary unmapped qcfail duplicate duplex duplex_forming status
SIRV1 control_rep1 12 12 0 0 0 0 0 0 0 Mapped
SIRV2 control_rep1 5 5 0 0 0 0 0 0 0 Mapped
SIRV3 control_rep1 14 14 0 0 0 0 0 0 0 Mapped
SIRV4 control_rep1 11 11 0 0 0 0 0 0 0 Mapped
SIRV5 control_rep1 45 44 0 1 0 0 0 0 0 Mapped
SIRV6 control_rep1 107 104 0 3 0 0 0 0 0 Mapped
SIRV7 control_rep1 8 7 0 1 0 0 0 0 0 Mapped
* control_rep1 3 0 0 0 3 0 0 0 0 Unmapped
ref sample_name total primary secondary supplementary unmapped qcfail duplicate duplex duplex_forming status
SIRV1 control_rep2 12 12 0 0 0 0 0 0 0 Mapped
SIRV2 control_rep2 5 5 0 0 0 0 0 0 0 Mapped
SIRV3 control_rep2 14 14 0 0 0 0 0 0 0 Mapped
SIRV4 control_rep2 11 11 0 0 0 0 0 0 0 Mapped
SIRV5 control_rep2 45 44 0 1 0 0 0 0 0 Mapped
SIRV6 control_rep2 107 104 0 3 0 0 0 0 0 Mapped
SIRV7 control_rep2 8 7 0 1 0 0 0 0 0 Mapped
* control_rep2 3 0 0 0 3 0 0 0 0 Unmapped
ref sample_name total primary secondary supplementary unmapped qcfail duplicate duplex duplex_forming status
SIRV1 treated_rep1 16 16 0 0 0 0 0 0 0 Mapped
SIRV2 treated_rep1 7 7 0 0 0 0 0 0 0 Mapped
SIRV3 treated_rep1 12 12 0 0 0 0 0 0 0 Mapped
SIRV4 treated_rep1 14 14 0 0 0 0 0 0 0 Mapped
SIRV5 treated_rep1 33 33 0 0 0 0 0 0 0 Mapped
SIRV6 treated_rep1 112 110 0 2 0 0 0 0 0 Mapped
SIRV7 treated_rep1 8 8 0 0 0 0 0 0 0 Mapped
* treated_rep1 0 0 0 0 0 0 0 0 0 Unmapped
ref sample_name total primary secondary supplementary unmapped qcfail duplicate duplex duplex_forming status
SIRV1 treated_rep2 16 16 0 0 0 0 0 0 0 Mapped
SIRV2 treated_rep2 7 7 0 0 0 0 0 0 0 Mapped
SIRV3 treated_rep2 12 12 0 0 0 0 0 0 0 Mapped
SIRV4 treated_rep2 14 14 0 0 0 0 0 0 0 Mapped
SIRV5 treated_rep2 33 33 0 0 0 0 0 0 0 Mapped
SIRV6 treated_rep2 112 110 0 2 0 0 0 0 0 Mapped
SIRV7 treated_rep2 8 8 0 0 0 0 0 0 0 Mapped
* treated_rep2 0 0 0 0 0 0 0 0 0 Unmapped

Sample metadata

Metadata for each input sample as parsed from the sample sheet and workflow parameters. Includes the sample alias, barcode, type, and any experimental design columns such as condition or batch that were supplied for differential analysis.

Field Value
barcode barcode01
type test_sample
run_ids ['8c239806e6f576cd17d6b7d532976b1fe830f9c6']
basecall_models []
alias control_rep1
condition control
batch b1
n_primary 197
n_unmapped 3
src_xam None
src_xai None
has_stats True
Field Value
barcode barcode02
type test_sample
run_ids ['8c239806e6f576cd17d6b7d532976b1fe830f9c6']
basecall_models []
alias control_rep2
condition control
batch b2
n_primary 197
n_unmapped 3
src_xam None
src_xai None
has_stats True
Field Value
barcode barcode03
type test_sample
run_ids ['8c239806e6f576cd17d6b7d532976b1fe830f9c6']
basecall_models []
alias treated_rep1
condition treated
batch b1
n_primary 200
n_unmapped 0
src_xam None
src_xai None
has_stats True
Field Value
barcode barcode04
type test_sample
run_ids ['8c239806e6f576cd17d6b7d532976b1fe830f9c6']
basecall_models []
alias treated_rep2
condition treated
batch b2
n_primary 200
n_unmapped 0
src_xam None
src_xai None
has_stats True

Reference and Annotation Checks

Results of compatibility checks between the supplied reference genome and annotation performed before bambu transcript modelling. Build and provider hints are inferred from sequence names and file content to help identify mismatched genome/annotation combinations.

Check Value
Overlapping seqnames 7
Seqnames only in annotation 0
Seqnames only in reference 0
Annotation records retained 354
Unstranded records excluded 0
Annotation attributes sanitised 0

Build and Provider Hints

Evidence Hints
Reference build None detected
Annotation build None detected
Reference provider None detected
Annotation provider None detected

Bambu Quality Control

Quality metrics from the bambu transcript discovery and quantification run. Library size statistics show cohort-level summary values to flag large inter-sample variation that could affect CPM normalisation, with the individual per-sample counts listed below. The transcript discovery table shows the transcriptome mode used, the novel discovery rate (NDR) threshold applied, and how many transcripts were present before and after low-count filtering.

Library Size Statistics

Metric Value
Samples analyzed 4
Median library size 162 reads
Min library size 161 reads
Max library size 162 reads
Library size ratio (max/min) 1.01x

Transcript Discovery

Metric Value
Transcriptome mode discover
NDR used automatic
Transcripts before filtering 68
Transcripts after filtering 52
Transcripts removed 16
Median transcripts per sample 45.5
Unique genes (after filter) 7

Per-Sample Library Sizes

Sample Library Size
treated_rep2 162
treated_rep1 162
control_rep2 161
control_rep1 161

Cohort transcriptome

Summary of the joint cohort bambu transcriptome model. Shows the total number of transcripts and genes in the final model, and the top 500 rows of the transcript abundance count table.

Metric Value
Transcripts 52
Genes 7

Top 500 most abundant transcripts

TXNAME GENEID NDR novelGene novelTranscript txClassDescription readCount relReadCount relSubsetCount txid eqClassById control_rep1 control_rep2 treated_rep1 treated_rep2
SIRV602 SIRV6 nan False False annotation nan nan nan 45 45 21.67851 21.67851 15.31415 15.31415
SIRV609 SIRV6 nan False False annotation nan nan nan 52 47;48;52;55 13.0 13.0 20.0 20.0
SIRV614 SIRV6 nan False False annotation nan nan nan 57 57 9.0 9.0 9.0 9.0
SIRV615 SIRV6 nan False False annotation nan nan nan 58 44;45;51;55;56;58 6.72946 6.72946 10.96323 10.96323
SIRV506 SIRV5 nan False False annotation nan nan nan 37 32;33;36;37;39;41 9.16075 9.16075 7.73527 7.73527
SIRV605 SIRV6 nan False False annotation nan nan nan 48 48;55 5.0 5.0 8.71205 8.71205
SIRV507 SIRV5 nan False False annotation nan nan nan 38 38 7.0 7.0 6.0 6.0
SIRV109 SIRV1 nan False False annotation nan nan nan 7 7 5.0 5.0 6.0 6.0
SIRV606 SIRV6 nan False False annotation nan nan nan 49 44;47;48;49;53;55 3.85266 3.85266 5.48335 5.48335
SIRV406 SIRV4 nan False False annotation nan nan nan 28 28 4.0 4.0 4.0 4.0
SIRV607 SIRV6 nan False False annotation nan nan nan 50 50 5.0 5.0 3.0 3.0
SIRV616 SIRV6 nan False False annotation nan nan nan 59 59 4.14734 4.14734 3.51665 3.51665
SIRV608 SIRV6 nan False False annotation nan nan nan 51 44;45;51;55 4.3357 4.3357 3.2816 3.2816
SIRV512 SIRV5 nan False False annotation nan nan nan 43 43 4.0 4.0 3.0 3.0
SIRV604 SIRV6 nan False False annotation nan nan nan 47 47 3.19224 3.19224 3.52309 3.52309
SIRV505 SIRV5 nan False False annotation nan nan nan 36 36 3.54679 3.54679 2.78507 2.78507
SIRV702 SIRV7 nan False False annotation nan nan nan 63 63 3.0 3.0 3.0 3.0
SIRV610 SIRV6 nan False False annotation nan nan nan 53 53 3.0 3.0 3.0 3.0
SIRV403 SIRV4 nan False False annotation nan nan nan 25 25 4.0 4.0 2.0 2.0
SIRV703 SIRV7 nan False False annotation nan nan nan 64 64 3.0 3.0 3.0 3.0
SIRV202 SIRV2 nan False False annotation nan nan nan 9 9 1.33333 1.33333 4.0 4.0
SIRV410 SIRV4 nan False False annotation nan nan nan 31 31 1.0 1.0 4.0 4.0
SIRV105 SIRV1 nan False False annotation nan nan nan 4 4 2.0 2.0 3.0 3.0
SIRV305 SIRV3 nan False False annotation nan nan nan 18 18 2.49677 2.49677 2.0 2.0
SIRV309 SIRV3 nan False False annotation nan nan nan 22 22 2.0 2.0 2.0 2.0
SIRV310 SIRV3 nan False False annotation nan nan nan 23 23 3.0 3.0 1.0 1.0
SIRV302 SIRV3 nan False False annotation nan nan nan 15 15 1.54708 1.54708 2.0 2.0
SIRV502 SIRV5 nan False False annotation nan nan nan 33 33 2.24965 2.24965 1.28899 1.28899
SIRV103 SIRV1 nan False False annotation nan nan nan 3 3 1.0 1.0 2.0 2.0
SIRV102 SIRV1 nan False False annotation nan nan nan 2 2 1.0 1.0 2.0 2.0
SIRV308 SIRV3 nan False False annotation nan nan nan 21 21 1.0 1.0 2.0 2.0
SIRV408 SIRV4 nan False False annotation nan nan nan 29 29 1.0 1.0 2.0 2.0
SIRV201 SIRV2 nan False False annotation nan nan nan 8 8 2.66667 2.66667 0.0 0.0
SIRV510 SIRV5 nan False False annotation nan nan nan 41 41 1.08609 1.08609 1.19068 1.19068
SIRV601 SIRV6 nan False False annotation nan nan nan 44 44 1.06408 1.06408 1.07152 1.07152
SIRV603 SIRV6 nan False False annotation nan nan nan 46 46;53 1.0 1.0 1.13435 1.13435
SIRV704 SIRV7 nan False False annotation nan nan nan 65 65 2.0 2.0 0.0 0.0
SIRV617 SIRV6 nan False False annotation nan nan nan 60 60 0.0 0.0 2.0 2.0
SIRV404 SIRV4 nan False False annotation nan nan nan 26 26 1.0 1.0 1.0 1.0
SIRV504 SIRV5 nan False False annotation nan nan nan 35 35 1.0 1.0 1.0 1.0
SIRV203 SIRV2 nan False False annotation nan nan nan 10 10 0.0 0.0 2.0 2.0
SIRV303 SIRV3 nan False False annotation nan nan nan 16 14;16 1.70776 1.70776 0.0 0.0
SIRV511 SIRV5 nan False False annotation nan nan nan 42 42 1.54754 1.54754 0.0 0.0
SIRV307 SIRV3 nan False False annotation nan nan nan 20 20 1.24839 1.24839 0.0 0.0
SIRV509 SIRV5 nan False False annotation nan nan nan 40 40 1.22693 1.22693 0.0 0.0
SIRV508 SIRV5 nan False False annotation nan nan nan 39 39 1.18226 1.18226 0.0 0.0
SIRV101 SIRV1 nan False False annotation nan nan nan 1 1 1.0 1.0 0.0 0.0
SIRV204 SIRV2 nan False False annotation nan nan nan 11 11 0.5 0.5 0.5 0.5
SIRV206 SIRV2 nan False False annotation nan nan nan 13 13 0.5 0.5 0.5 0.5
SIRV611 SIRV6 nan False False annotation nan nan nan 54 54 1.0 1.0 0.0 0.0
SIRV706 SIRV7 nan False False annotation nan nan nan 67 67 0.0 0.0 0.5 0.5
SIRV708 SIRV7 nan False False annotation nan nan nan 68 68 0.0 0.0 0.5 0.5

Per-sample transcriptomes

Per-sample transcript and gene count summaries derived from the individual bambu quantification runs. Each tab shows the number of transcripts and genes detected in that sample after filtering. These can be used to spot samples with unusually low transcript detection compared to the rest of the cohort.

Metric Value
Transcripts 48
Genes 7
Metric Value
Transcripts 48
Genes 7
Metric Value
Transcripts 43
Genes 7
Metric Value
Transcripts 43
Genes 7

SQANTI3 classification

Structural classification of transcript isoforms by SQANTI3. Each transcript is assigned a category based on how its splice junctions and exon structure compared to the reference annotation. The table shows the count of transcripts in each category per sample and for the whole cohort.

Sample Full splice match Incomplete splice match Novel in catalog Novel not in catalog Antisense Genic intron Genic Intergenic
control_rep1 48 0 0 0 0 0 0 0
control_rep2 48 0 0 0 0 0 0 0
treated_rep1 43 0 0 0 0 0 0 0
treated_rep2 43 0 0 0 0 0 0 0
cohort 52 0 0 0 0 0 0 0

Full splice match: Reference and query isoforms have the same number of exons and all internal junctions agree.
 Incomplete splice match: Query isoform has fewer 5′ exons than the reference, with matching internal junctions.
 Novel in catalog: No full or incomplete splice match, but uses a combination of known donor/acceptor splice sites.
 Novel not in catalog: No full or incomplete splice match, with at least one unannotated donor or acceptor splice site.
 Antisense: No same-strand reference overlap, but antisense to an annotated gene.
 Genic intron: Query isoform is fully contained within an annotated intron.
 Genic: Query isoform overlaps introns and exons.
 Intergenic: Query isoform lies in an intergenic region.

Differential Analysis Quality Control

Quality control summary for the differential expression analyses. Shows the experimental design and the number of samples per group. For each contrast, the statistical methods chosen by DESeq2 and DEXSeq are reported , including the dispersion estimation strategy (parametric or gene-wise fallback) and size factor normalisation method, alongside the analysis status and count of significant hits. Any warnings about low sample numbers, dispersion fallbacks, dropped covariates, or analysis failures are highlighted here.

⚠️ Sample Size Warning: Some groups have n<3 (recommended minimum). Underpowered designs may have reduced statistical power and increased false negative rate.
⚠️ Gene-wise dispersion fallback used (reduced power). DESeq2: condition_treated_vs_control
❌ DTU Analysis Failed: 1 contrast(s) could not perform DTU testing. Affected: condition_treated_vs_control. See DTU_ANALYSIS_FAILED.txt files for details.

Experimental Design

Parameter Value
Total samples 4
Condition column condition
Reference level control
Covariates batch
Number of contrasts 1

Sample Sizes per Group

Group Samples Status Note
control 2 ⚠️ Low power
treated 2 ⚠️ Low power

Statistical Methods & Warnings

Contrast DESeq2 size factors DESeq2 dispersion DEXSeq size factors DEXSeq dispersion DEXSeq covariates dropped DGE status DTU status
condition_treated_vs_control ratio gene-wise (fallback) ratio parametric none SUCCESS FAILED

Results Summary by Contrast

Contrast Samples DGE Status DGE Significant (FDR<0.05) DGE Up DGE Down DTU Status DTU Genes (q<0.05)
condition_treated_vs_control 2 vs 2 SUCCESS 0 0 0 FAILED N/A

Quality Warnings Summary

Warning Type Details
Sample Size Some groups have n<3 (recommended minimum)
Gene-wise Dispersion Fallback DESeq2 (1 contrasts)
DTU Failure 1 contrasts failed

Differential gene expression

Differential gene expression results from DESeq2. The heatmap, PCA plot, and sample distance matrix are derived from CPM-normalised counts and give an overview of sample clustering relative to the experimental conditions. Each contrast tab shows a results table of genes ranked by adjusted p-value and a volcano plot highlighting significantly up- and down-regulated genes.


(Left) Hierarchical clustering heatmap of the top 150 most variable gene across all samples. Rows represent gene (Z-scored and log2 transformed fold changes), columns represent samples. Dendrograms show clustering of both genes and samples. (Middle) Principal component analysis (PCA showing the first two principal components) of sample gene expression profiles. Each point represents a sample, coloured by condition. Samples that cluster together have similar overall expression profiles. (Right) Sample-to-sample Euclidean distance matrix calculated from log2-transformed fold change values. Lower values (darker blue) indicate more similar expression profiles between samples.

Note: DTU analysis may be underpowered (n=4, recommend n>=6 with >=3 per group)

GENEID newGeneClass baseMean log2FoldChange lfcSE stat pvalue padj
SIRV5 annotation 33.666 -0.558 0.253 -2.209 0.027 0.190
SIRV1 annotation 13.464 0.242 0.395 0.612 0.541 0.817
SIRV2 annotation 5.975 0.405 0.597 0.678 0.497 0.817
SIRV3 annotation 12.519 -0.196 0.408 -0.479 0.632 0.817
SIRV4 annotation 11.977 0.161 0.418 0.385 0.700 0.817
SIRV7 annotation 7.517 -0.273 0.528 -0.517 0.605 0.817
SIRV6 annotation 107.958 6.694e-08 0.139 4.820e-07 1.000 1.000
Table showing the top 500 genes sorted by adjusted p-value.


Gene expression volcano Plot

Differential transcript usage

Differential transcript usage results from DEXSeq. Unlike DGE, DTU tests whether individual transcripts change their proportional contribution to total gene expression between conditions , i.e. isoform switching , rather than testing for changes in total gene abundance. The heatmap, PCA, and sample distance matrix use CPM-normalised counts. Each contrast tab shows a results table ranked by adjusted p-value and a volcano plot.


(Left) Hierarchical clustering heatmap of the top 150 most variable transcript across all samples. Rows represent transcript (Z-scored and log2 transformed fold changes), columns represent samples. Dendrograms show clustering of both genes and samples. (Middle) Principal component analysis (PCA showing the first two principal components) of sample transcript expression profiles. Each point represents a sample, coloured by condition. Samples that cluster together have similar overall expression profiles. (Right) Sample-to-sample Euclidean distance matrix calculated from log2-transformed fold change values. Lower values (darker blue) indicate more similar expression profiles between samples.
❌ DTU analysis failed for this contrast. See condition_treated_vs_control/DTU_ANALYSIS_FAILED.txt for detailed explanation.

Empty results indicate analysis failure, not 'no DTU detected'.

No DTU results available for this contrast.

Software versions

Name Version
minimap2 2.28-r1209
samtools 1.23.1
gffread 0.12.9
bambu 3.12.1
DESeq2 1.50.2
DEXSeq 1.56.0
SQANTI3 5.5.1
modkit 0.6.3

Workflow parameters

Key Value
fastq test_data/smoke/de
bam None
sample None
sample_sheet test_data/smoke/sample_sheet_de.csv
out_dir wf-transcriptomes
igv False
ref_genome test_data/smoke/reference.fa
ref_annotation test_data/smoke/annotation.gtf
transcriptome_mode discover
direct_rna False
de_analysis True
condition_column condition
covariates batch
reference_level control
analyse_unclassified False
analyse_fail False
mod_codes None
force_alignment False
ndr None
sqanti_skip_orf True
store_dir wf-transcriptomes/store_dir