PTox-Production-phase2-Drosophila-include-dups
A modular tool to aggregate results from bioinformatics analyses across many samples into a single report.
This report has been generated by the nf-core/rnaseq analysis pipeline. For information about how to interpret these results, please see the documentation.
/users/project/precisiontox_008279_no_backup/production_phase2_data_dec2025/analyse_duplicated_data/rnaseq_run-Drosophila/work/ae/1e1c781747aefe456128fbaddb471f
General Statistics
| Sample Name | dupInt | Duplication | 5'-3' bias | M Aligned | Exonic | Intronic | Intergenic | Overlapping Exon | Error rate | Non-primary | Reads mapped | % Mapped | % Proper pairs | % MapQ 0 reads | Total seqs | Mean insert | Reads | Reads mapped | % Reads mapped |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| FAH000ZA1 | 0.4 | 82.8% | 0.43 | 4.5M | 3.5M | 0.3M | 0.2M | 0.6M | 0.72% | 1.3M | 4.5M | 100.0% | 0.0% | 2.0% | 4.5M | 0.0bp | 5.8M | 5.8M | 100.0% |
| FAH000ZA2 | 0.5 | 83.0% | 0.46 | 4.5M | 3.5M | 0.4M | 0.2M | 0.6M | 0.72% | 1.2M | 4.5M | 100.0% | 0.0% | 1.9% | 4.5M | 0.0bp | 5.7M | 5.7M | 100.0% |
| FAH000ZA3 | 0.7 | 87.2% | 0.39 | 5.4M | 4.2M | 0.4M | 0.3M | 0.7M | 0.74% | 1.5M | 5.4M | 100.0% | 0.0% | 2.2% | 5.4M | 0.0bp | 6.8M | 6.8M | 100.0% |
| FAH000ZA4 | 0.5 | 84.2% | 0.44 | 4.6M | 3.6M | 0.4M | 0.2M | 0.6M | 0.70% | 1.5M | 4.6M | 100.0% | 0.0% | 2.4% | 4.6M | 0.0bp | 6.1M | 6.1M | 100.0% |
| FAH000ZA5 | 0.8 | 88.1% | 0.42 | 6.0M | 4.5M | 0.6M | 0.3M | 0.8M | 0.70% | 1.8M | 6.0M | 100.0% | 0.0% | 2.9% | 6.0M | 0.0bp | 7.8M | 7.8M | 100.0% |
| FAH000ZA6 | 0.5 | 83.4% | 0.44 | 5.1M | 3.9M | 0.4M | 0.3M | 0.7M | 0.71% | 1.6M | 5.1M | 100.0% | 0.0% | 2.4% | 5.1M | 0.0bp | 6.7M | 6.7M | 100.0% |
| FAH000ZA7 | 0.7 | 86.3% | 0.46 | 5.5M | 4.2M | 0.5M | 0.3M | 0.8M | 0.67% | 1.4M | 5.5M | 100.0% | 0.0% | 2.4% | 5.5M | 0.0bp | 6.9M | 6.9M | 100.0% |
| FAH000ZA8 | 0.4 | 81.0% | 0.41 | 4.4M | 3.5M | 0.4M | 0.2M | 0.6M | 0.71% | 1.1M | 4.4M | 100.0% | 0.0% | 1.6% | 4.4M | 0.0bp | 5.5M | 5.5M | 100.0% |
| FAH000ZB1 | 0.5 | 83.9% | 0.41 | 5.7M | 4.4M | 0.5M | 0.3M | 0.8M | 0.68% | 1.3M | 5.7M | 100.0% | 0.0% | 1.7% | 5.7M | 0.0bp | 7.0M | 7.0M | 100.0% |
| FAH000ZB2 | 0.5 | 85.1% | 0.40 | 6.6M | 5.0M | 0.5M | 0.3M | 0.9M | 0.70% | 2.0M | 6.6M | 100.0% | 0.0% | 2.4% | 6.6M | 0.0bp | 8.6M | 8.6M | 100.0% |
| FAH000ZB3 | 0.8 | 88.1% | 0.45 | 6.6M | 5.1M | 0.5M | 0.3M | 0.9M | 0.73% | 2.0M | 6.6M | 100.0% | 0.0% | 2.2% | 6.6M | 0.0bp | 8.5M | 8.5M | 100.0% |
| FAH000ZB4 | 0.5 | 81.9% | 0.46 | 4.2M | 3.2M | 0.3M | 0.2M | 0.5M | 0.73% | 1.2M | 4.2M | 100.0% | 0.0% | 2.3% | 4.2M | 0.0bp | 5.4M | 5.4M | 100.0% |
| FAH000ZB5 | 0.6 | 85.3% | 0.38 | 5.8M | 4.4M | 0.5M | 0.3M | 0.7M | 0.70% | 1.9M | 5.8M | 100.0% | 0.0% | 2.7% | 5.8M | 0.0bp | 7.7M | 7.7M | 100.0% |
| FAH000ZB6 | 0.4 | 79.8% | 0.40 | 3.5M | 2.7M | 0.3M | 0.2M | 0.5M | 0.73% | 0.9M | 3.5M | 100.0% | 0.0% | 2.0% | 3.5M | 0.0bp | 4.5M | 4.5M | 100.0% |
| FAH000ZB7 | 0.6 | 85.4% | 0.43 | 5.4M | 4.3M | 0.4M | 0.3M | 0.7M | 0.70% | 1.4M | 5.4M | 100.0% | 0.0% | 1.8% | 5.4M | 0.0bp | 6.8M | 6.8M | 100.0% |
| FAH000ZB8 | 0.3 | 79.7% | 0.38 | 4.2M | 3.2M | 0.3M | 0.2M | 0.5M | 0.72% | 1.3M | 4.2M | 100.0% | 0.0% | 2.4% | 4.2M | 0.0bp | 5.5M | 5.5M | 100.0% |
| FAH007LA1 | 0.7 | 86.2% | 0.39 | 5.6M | 4.3M | 0.5M | 0.3M | 0.8M | 0.68% | 1.7M | 5.6M | 100.0% | 0.0% | 2.8% | 5.6M | 0.0bp | 7.4M | 7.4M | 100.0% |
| FAH007LA2 | 0.5 | 82.6% | 0.43 | 4.6M | 3.6M | 0.4M | 0.2M | 0.6M | 0.71% | 1.0M | 4.6M | 100.0% | 0.0% | 1.5% | 4.6M | 0.0bp | 5.6M | 5.6M | 100.0% |
| FAH007LA3 | 0.7 | 86.5% | 0.45 | 5.8M | 4.4M | 0.5M | 0.3M | 0.8M | 0.70% | 1.5M | 5.8M | 100.0% | 0.0% | 2.1% | 5.8M | 0.0bp | 7.3M | 7.3M | 100.0% |
| FAH007LA4 | 0.5 | 82.7% | 0.43 | 4.4M | 3.4M | 0.4M | 0.2M | 0.6M | 0.75% | 1.1M | 4.4M | 100.0% | 0.0% | 1.9% | 4.4M | 0.0bp | 5.5M | 5.5M | 100.0% |
| FAH007LB1 | 0.5 | 84.0% | 0.45 | 5.0M | 3.9M | 0.4M | 0.2M | 0.7M | 0.71% | 1.2M | 5.0M | 100.0% | 0.0% | 1.8% | 5.0M | 0.0bp | 6.3M | 6.3M | 100.0% |
| FAH007LB2 | 0.6 | 85.7% | 0.40 | 6.1M | 4.7M | 0.5M | 0.3M | 0.8M | 0.69% | 1.4M | 6.1M | 100.0% | 0.0% | 1.7% | 6.1M | 0.0bp | 7.5M | 7.5M | 100.0% |
| FAH007LB3 | 0.6 | 83.4% | 0.41 | 4.6M | 3.5M | 0.4M | 0.2M | 0.6M | 0.71% | 1.2M | 4.6M | 100.0% | 0.0% | 2.0% | 4.6M | 0.0bp | 5.7M | 5.7M | 100.0% |
| FAH007LB4 | 0.5 | 85.2% | 0.41 | 6.2M | 4.8M | 0.5M | 0.3M | 0.8M | 0.74% | 1.7M | 6.2M | 100.0% | 0.0% | 2.1% | 6.2M | 0.0bp | 7.9M | 7.9M | 100.0% |
| FAI000ZA1 | 0.6 | 85.3% | 0.41 | 6.0M | 4.6M | 0.5M | 0.3M | 0.7M | 0.73% | 1.7M | 6.0M | 100.0% | 0.0% | 2.0% | 6.0M | 0.0bp | 7.6M | 7.6M | 100.0% |
| FAI000ZA2 | 0.4 | 79.4% | 0.33 | 3.4M | 2.6M | 0.3M | 0.2M | 0.4M | 0.74% | 1.0M | 3.4M | 100.0% | 0.0% | 2.2% | 3.4M | 0.0bp | 4.4M | 4.4M | 100.0% |
| FAI000ZA3 | 0.6 | 86.7% | 0.38 | 8.5M | 6.6M | 0.7M | 0.4M | 1.1M | 0.74% | 2.4M | 8.5M | 100.0% | 0.0% | 2.0% | 8.5M | 0.0bp | 10.9M | 10.9M | 100.0% |
| FAI000ZA4 | 0.5 | 82.5% | 0.44 | 4.0M | 3.0M | 0.4M | 0.2M | 0.5M | 0.72% | 1.3M | 4.0M | 100.0% | 0.0% | 2.6% | 4.0M | 0.0bp | 5.2M | 5.2M | 100.0% |
| FAI000ZA5 | 0.5 | 83.6% | 0.39 | 4.2M | 3.2M | 0.4M | 0.2M | 0.5M | 0.81% | 1.4M | 4.2M | 100.0% | 0.0% | 2.9% | 4.2M | 0.0bp | 5.7M | 5.7M | 100.0% |
| FAI000ZA6 | 0.5 | 84.7% | 0.39 | 6.2M | 4.9M | 0.5M | 0.3M | 0.7M | 0.78% | 1.7M | 6.2M | 100.0% | 0.0% | 2.1% | 6.2M | 0.0bp | 7.9M | 7.9M | 100.0% |
| FAI000ZA7 | 0.7 | 85.7% | 0.36 | 4.6M | 3.7M | 0.3M | 0.2M | 0.6M | 0.81% | 1.2M | 4.6M | 100.0% | 0.0% | 2.0% | 4.6M | 0.0bp | 5.9M | 5.9M | 100.0% |
| FAI000ZA8 | 0.5 | 86.7% | 0.40 | 8.6M | 6.7M | 0.7M | 0.4M | 1.1M | 0.74% | 2.4M | 8.6M | 100.0% | 0.0% | 2.0% | 8.6M | 0.0bp | 11.1M | 11.1M | 100.0% |
| FAI000ZB1 | 0.7 | 86.5% | 0.33 | 5.5M | 4.3M | 0.4M | 0.3M | 0.7M | 0.72% | 1.6M | 5.5M | 100.0% | 0.0% | 2.1% | 5.5M | 0.0bp | 7.1M | 7.1M | 100.0% |
| FAI000ZB2 | 0.5 | 83.5% | 0.33 | 4.9M | 3.8M | 0.4M | 0.3M | 0.6M | 0.76% | 1.9M | 4.9M | 100.0% | 0.0% | 3.6% | 4.9M | 0.0bp | 6.9M | 6.9M | 100.0% |
| FAI000ZB3 | 0.5 | 83.6% | 0.47 | 4.2M | 3.2M | 0.3M | 0.2M | 0.5M | 0.71% | 1.3M | 4.2M | 100.0% | 0.0% | 2.6% | 4.2M | 0.0bp | 5.5M | 5.5M | 100.0% |
| FAI000ZB4 | 0.5 | 85.5% | 0.43 | 6.1M | 4.7M | 0.4M | 0.3M | 0.7M | 0.75% | 1.7M | 6.1M | 100.0% | 0.0% | 2.2% | 6.1M | 0.0bp | 7.8M | 7.8M | 100.0% |
| FAI000ZB5 | 0.6 | 84.8% | 0.36 | 4.6M | 3.5M | 0.4M | 0.2M | 0.5M | 0.74% | 1.5M | 4.6M | 100.0% | 0.0% | 2.5% | 4.6M | 0.0bp | 6.0M | 6.0M | 100.0% |
| FAI000ZB6 | 0.6 | 85.9% | 0.36 | 5.9M | 4.7M | 0.4M | 0.3M | 0.7M | 0.77% | 1.5M | 5.9M | 100.0% | 0.0% | 1.8% | 5.9M | 0.0bp | 7.4M | 7.4M | 100.0% |
| FAI000ZB7 | 0.6 | 84.0% | 0.37 | 4.8M | 3.6M | 0.4M | 0.2M | 0.6M | 0.72% | 1.6M | 4.8M | 100.0% | 0.0% | 2.7% | 4.8M | 0.0bp | 6.4M | 6.4M | 100.0% |
| FAI000ZB8 | 0.8 | 86.6% | 0.31 | 4.2M | 3.3M | 0.3M | 0.2M | 0.5M | 0.78% | 1.2M | 4.2M | 100.0% | 0.0% | 2.2% | 4.2M | 0.0bp | 5.4M | 5.4M | 100.0% |
| FAI078AA1 | 0.5 | 84.5% | 0.40 | 5.1M | 4.0M | 0.4M | 0.2M | 0.6M | 0.75% | 1.4M | 5.1M | 100.0% | 0.0% | 2.0% | 5.1M | 0.0bp | 6.5M | 6.5M | 100.0% |
| FAI078AA2 | 0.6 | 87.6% | 0.42 | 7.5M | 5.8M | 0.6M | 0.4M | 0.9M | 0.73% | 2.0M | 7.5M | 100.0% | 0.0% | 1.9% | 7.5M | 0.0bp | 9.5M | 9.5M | 100.0% |
| FAI078AA3 | 0.5 | 79.9% | 0.47 | 2.4M | 1.9M | 0.2M | 0.1M | 0.3M | 0.73% | 0.7M | 2.4M | 100.0% | 0.0% | 2.2% | 2.4M | 0.0bp | 3.1M | 3.1M | 100.0% |
| FAI078AA4 | 0.4 | 77.9% | 0.42 | 2.9M | 2.2M | 0.2M | 0.1M | 0.4M | 0.72% | 0.7M | 2.9M | 100.0% | 0.0% | 1.9% | 2.9M | 0.0bp | 3.6M | 3.6M | 100.0% |
| FAI078AB1 | 0.5 | 83.4% | 0.43 | 4.8M | 3.8M | 0.4M | 0.2M | 0.6M | 0.74% | 1.0M | 4.8M | 100.0% | 0.0% | 1.4% | 4.8M | 0.0bp | 5.7M | 5.7M | 100.0% |
| FAI078AB2 | 0.5 | 82.5% | 0.44 | 3.8M | 3.0M | 0.3M | 0.2M | 0.5M | 0.74% | 1.1M | 3.8M | 100.0% | 0.0% | 2.1% | 3.8M | 0.0bp | 4.9M | 4.9M | 100.0% |
| FAI078AB3 | 0.6 | 87.1% | 0.39 | 7.2M | 5.6M | 0.5M | 0.3M | 0.9M | 0.74% | 1.9M | 7.2M | 100.0% | 0.0% | 1.9% | 7.2M | 0.0bp | 9.1M | 9.1M | 100.0% |
| FAI078AB4 | 0.4 | 80.4% | 0.46 | 3.3M | 2.6M | 0.2M | 0.2M | 0.4M | 0.75% | 0.9M | 3.3M | 100.0% | 0.0% | 2.1% | 3.3M | 0.0bp | 4.2M | 4.2M | 100.0% |
Sample status checks
Reports on sample strandedness status, and any failures in trimming or mapping.
Strandedness Checks
The strandedness used for analysis in this workflow can either be provided by the user or automatically inferred by Salmon using a sample of reads. In both cases, strandedness is verified at the end of the workflow using RSeQC's infer_experiment.py on genomic alignments. In this table, a pass indicates a match between the supplied strandedness (or that inferred by Salmon) and RSeQC results. A fail indicates a mismatch or 'undetermined' strandedness. 'Undetermined' strandedness can signal QC issues, including potential genomic DNA contamination.
Note: Rows are duplicated for an 'auto' setting to allow comparison of statistics between inference methods.
| Sample | Status | Strand inference method | Provided strandedness | Inferred strandedness | Sense (%) | Antisense (%) | Unstranded (%) |
|---|---|---|---|---|---|---|---|
| FAH000ZA1 | fail | RSeQC | auto | forward | 86.5 | 1.6 | 11.9 |
| FAH000ZA2 | fail | RSeQC | auto | forward | 86.3 | 1.7 | 11.9 |
| FAH000ZA3 | fail | RSeQC | auto | forward | 85.4 | 1.8 | 12.8 |
| FAH000ZA4 | fail | RSeQC | auto | forward | 86.0 | 1.8 | 12.2 |
| FAH000ZA5 | fail | RSeQC | auto | forward | 85.6 | 2.0 | 12.4 |
| FAH000ZA6 | fail | RSeQC | auto | forward | 86.1 | 1.8 | 12.1 |
| FAH000ZA7 | fail | RSeQC | auto | forward | 86.6 | 1.8 | 11.6 |
| FAH000ZA8 | fail | RSeQC | auto | forward | 86.4 | 1.6 | 12.0 |
| FAH000ZB1 | fail | RSeQC | auto | forward | 85.7 | 1.9 | 12.3 |
| FAH000ZB2 | fail | RSeQC | auto | forward | 85.6 | 2.1 | 12.2 |
| FAH000ZB3 | fail | RSeQC | auto | forward | 85.9 | 1.8 | 12.3 |
| FAH000ZB4 | fail | RSeQC | auto | forward | 86.0 | 1.8 | 12.2 |
| FAH000ZB5 | fail | RSeQC | auto | forward | 85.7 | 2.2 | 12.1 |
| FAH000ZB6 | fail | RSeQC | auto | forward | 86.3 | 1.7 | 11.9 |
| FAH000ZB7 | fail | RSeQC | auto | forward | 85.8 | 1.9 | 12.3 |
| FAH000ZB8 | fail | RSeQC | auto | forward | 85.5 | 2.0 | 12.4 |
| FAH007LA1 | fail | RSeQC | auto | forward | 86.4 | 2.0 | 11.6 |
| FAH007LA2 | fail | RSeQC | auto | forward | 86.3 | 1.6 | 12.1 |
| FAH007LA3 | fail | RSeQC | auto | forward | 86.2 | 1.8 | 12.0 |
| FAH007LA4 | fail | RSeQC | auto | forward | 86.1 | 2.0 | 11.9 |
| FAH007LB1 | fail | RSeQC | auto | forward | 86.4 | 1.6 | 12.0 |
| FAH007LB2 | fail | RSeQC | auto | forward | 86.3 | 1.9 | 11.8 |
| FAH007LB3 | fail | RSeQC | auto | forward | 86.9 | 1.8 | 11.3 |
| FAH007LB4 | fail | RSeQC | auto | forward | 86.2 | 2.0 | 11.8 |
| FAI000ZA1 | fail | RSeQC | auto | forward | 86.6 | 1.7 | 11.7 |
| FAI000ZA2 | fail | RSeQC | auto | forward | 86.7 | 1.5 | 11.8 |
| FAI000ZA3 | fail | RSeQC | auto | forward | 86.3 | 1.9 | 11.7 |
| FAI000ZA4 | fail | RSeQC | auto | forward | 86.5 | 1.7 | 11.8 |
| FAI000ZA5 | fail | RSeQC | auto | forward | 85.9 | 1.9 | 12.1 |
| FAI000ZA6 | fail | RSeQC | auto | forward | 86.6 | 1.8 | 11.5 |
| FAI000ZA7 | fail | RSeQC | auto | forward | 86.3 | 1.5 | 12.2 |
| FAI000ZA8 | fail | RSeQC | auto | forward | 86.3 | 1.9 | 11.8 |
| FAI000ZB1 | fail | RSeQC | auto | forward | 86.4 | 1.5 | 12.0 |
| FAI000ZB2 | fail | RSeQC | auto | forward | 85.8 | 2.2 | 12.0 |
| FAI000ZB3 | fail | RSeQC | auto | forward | 86.0 | 1.7 | 12.3 |
| FAI000ZB4 | fail | RSeQC | auto | forward | 85.9 | 1.9 | 12.2 |
| FAI000ZB5 | fail | RSeQC | auto | forward | 86.2 | 1.7 | 12.2 |
| FAI000ZB6 | fail | RSeQC | auto | forward | 86.0 | 1.6 | 12.3 |
| FAI000ZB7 | fail | RSeQC | auto | forward | 86.5 | 1.7 | 11.8 |
| FAI000ZB8 | fail | RSeQC | auto | forward | 86.4 | 1.6 | 12.0 |
| FAI078AA1 | fail | RSeQC | auto | forward | 86.6 | 1.7 | 11.7 |
| FAI078AA2 | fail | RSeQC | auto | forward | 86.3 | 1.8 | 12.0 |
| FAI078AA3 | fail | RSeQC | auto | forward | 86.4 | 1.5 | 12.1 |
| FAI078AA4 | fail | RSeQC | auto | forward | 86.7 | 1.4 | 11.9 |
| FAI078AB1 | fail | RSeQC | auto | forward | 86.4 | 1.5 | 12.1 |
| FAI078AB2 | fail | RSeQC | auto | forward | 86.2 | 1.5 | 12.3 |
| FAI078AB3 | fail | RSeQC | auto | forward | 86.0 | 1.8 | 12.2 |
| FAI078AB4 | fail | RSeQC | auto | forward | 86.2 | 1.5 | 12.3 |
DupRadar
DupRadar provides duplication rate quality control for RNA-Seq datasets. Highly expressed genes can be expected to have a lot of duplicate reads, but high numbers of duplicates at low read counts can indicate low library complexity with technical duplication. This plot shows the general linear models - a summary of the gene duplication distributions.bioconductor.org/packages/release/bioc/html/dupRadar.html
Picard
Tools for manipulating high-throughput sequencing data.http://broadinstitute.github.io/picard
Mark Duplicates
Number of reads, categorised by duplication state. Pair counts are doubled - see help text for details.
The table in the Picard metrics file contains some columns referring read pairs and some referring to single reads.
To make the numbers in this plot sum correctly, values referring to pairs are doubled according to the scheme below:
READS_IN_DUPLICATE_PAIRS = 2 * READ_PAIR_DUPLICATESREADS_IN_UNIQUE_PAIRS = 2 * (READ_PAIRS_EXAMINED - READ_PAIR_DUPLICATES)READS_IN_UNIQUE_UNPAIRED = UNPAIRED_READS_EXAMINED - UNPAIRED_READ_DUPLICATESREADS_IN_DUPLICATE_PAIRS_OPTICAL = 2 * READ_PAIR_OPTICAL_DUPLICATESREADS_IN_DUPLICATE_PAIRS_NONOPTICAL = READS_IN_DUPLICATE_PAIRS - READS_IN_DUPLICATE_PAIRS_OPTICALREADS_IN_DUPLICATE_UNPAIRED = UNPAIRED_READ_DUPLICATESREADS_UNMAPPED = UNMAPPED_READS
Preseq
Estimates library complexity, showing how many additional unique reads are sequenced for increasing total read count.http://smithlabresearch.org/software/preseqDOI: 10.1038/nmeth.2375
A shallow curve indicates complexity saturation. The dashed line shows a perfectly complex library where total reads = unique reads.Complexity curve (molecule count)
Note that the x-axis is trimmed at the point where all the datasets show 80% of their maximum y-value, to avoid ridiculous scales.
QualiMap
Quality control of alignment data and its derivatives like feature counts.http://qualimap.bioinfo.cipf.esDOI: 10.1093/bioinformatics/btv566; 10.1093/bioinformatics/bts503
Genomic origin of reads
Classification of mapped reads as originating in exonic, intronic or intergenic regions. These can be displayed as either the number or percentage of mapped reads.
There are currently three main approaches to map reads to transcripts in an RNA-seq experiment: mapping reads to a reference genome to identify expressed transcripts that are annotated (and discover those that are unknown), mapping reads to a reference transcriptome, and de novo assembly of transcript sequences (Conesa et al. 2016).
For RNA-seq QC analysis, QualiMap can be used to assess alignments produced by the first of these approaches. For input, it requires a GTF annotation file along with a reference genome, which can be used to reconstruct the exon structure of known transcripts. This allows mapped reads to be grouped by whether they originate in an exonic region (for QualiMap, this may include 5′ and 3′ UTR regions as well as protein-coding exons), an intron, or an intergenic region (see the Qualimap 2 documentation).
The inferred genomic origins of RNA-seq reads are presented here as a bar graph showing either the number or percentage of mapped reads in each read dataset that have been assigned to each type of genomic region. This graph can be used to assess the proportion of useful reads in an RNA-seq experiment. That proportion can be reduced by the presence of intron sequences, especially if depletion of ribosomal RNA was used during sample preparation (Sims et al. 2014). It can also be reduced by off-target transcripts, which are detected in greater numbers at the sequencing depths needed to detect poorly-expressed transcripts (Tarazona et al. 2011).
Gene Coverage Profile
Mean distribution of coverage depth across the length of all mapped transcripts.
There are currently three main approaches to map reads to transcripts in an RNA-seq experiment: mapping reads to a reference genome to identify expressed transcripts that are annotated (and discover those that are unknown), mapping reads to a reference transcriptome, and de novo assembly of transcript sequences (Conesa et al. 2016).
For RNA-seq QC analysis, QualiMap can be used to assess alignments produced by the first of these approaches. For input, it requires a GTF annotation file along with a reference genome, which can be used to reconstruct the exon structure of known transcripts. QualiMap uses this information to calculate the depth of coverage along the length of each annotated transcript. For a set of reads mapped to a transcript, the depth of coverage at a given base position is the number of high-quality reads that map to the transcript at that position (Sims et al. 2014).
QualiMap calculates coverage depth at every base position of each annotated transcript. To enable meaningful comparison between transcripts, base positions are rescaled to relative positions expressed as percentage distance along each transcript (0%, 1%, …, 99%). For the set of transcripts with at least one mapped read, QualiMap plots the cumulative mapped-read depth (y-axis) at each relative transcript position (x-axis). This plot shows the gene coverage profile across all mapped transcripts for each read dataset. It provides a visual way to assess positional biases, such as an accumulation of mapped reads at the 3′ end of transcripts, which may indicate poor RNA quality in the original sample (Conesa et al. 2016).
The Normalised plot is calculated by MultiQC to enable comparison of samples with varying sequencing depth. The cumulative mapped-read depth at each position across the averaged transcript position are divided by the total for that sample across the entire averaged transcript.
RSeQC
Evaluates high throughput RNA-seq data.http://rseqc.sourceforge.netDOI: 10.1093/bioinformatics/bts356
Read Distribution
Read Distribution calculates how mapped reads are distributed over genome features.
Read Duplication
read_duplication.py calculates how many alignment positions have a certain number of exact duplicates. Note - plot truncated at 500 occurrences and binned.
Junction Annotation
Junction annotation compares detected splice junctions to a reference gene model. An RNA read can be spliced 2 or more times, each time is called a splicing event.
Junction Saturation
Junction Saturation counts the number of known splicing junctions that are observed in each dataset. If sequencing depth is sufficient, all (annotated) splice junctions should be rediscovered, resulting in a curve that reaches a plateau. Missing low abundance splice junctions can affect downstream analysis.
Infer experiment
Infer experiment counts the percentage of reads and read pairs that match the strandedness of overlapping transcripts. It can be used to infer whether RNA-seq library preps are stranded (sense or antisense).
Bam Stat
All numbers reported in millions.
Samtools
Toolkit for interacting with BAM/CRAM files.http://www.htslib.orgDOI: 10.1093/bioinformatics/btp352
Percent mapped
Alignment metrics from samtools stats; mapped vs. unmapped reads vs. reads mapped with MQ0.
For a set of samples that have come from the same multiplexed library, similar numbers of reads for each sample are expected. Large differences in numbers might indicate issues during the library preparation process. Whilst large differences in read numbers may be controlled for in downstream processings (e.g. read count normalisation), you may wish to consider whether the read depths achieved have fallen below recommended levels depending on the applications.
Low alignment rates could indicate contamination of samples (e.g. adapter sequences), low sequencing quality or other artefacts. These can be further investigated in the sequence level QC (e.g. from FastQC).
Reads mapped with MQ0 often indicate that the reads are ambiguously mapped to multiple locations in the reference sequence. This can be due to repetitive regions in the genome, the presence of alternative contigs in the reference, or due to reads that are too short to be uniquely mapped. These reads are often filtered out in downstream analyses.
Alignment stats
This module parses the output from samtools stats. All numbers in millions.
Flagstat
This module parses the output from samtools flagstat
Flagstat: Percentage of total
This module parses the output from samtools flagstat
Mapped reads per contig
The samtools idxstats tool counts the number of mapped reads per chromosome / contig. Chromosomes with < 0.1% of the total aligned reads are omitted from this plot.
Sample relationships
Plots interrogating sample relationships, based on final count matrices.
STAR_SALMON DESeq2 sample similarity
is generated from clustering by Euclidean distances between DESeq2 rlog values for each sample in the deseq2_qc.r script.
STAR_SALMON DESeq2 PCA plot
PCA plot between samples in the experiment. These values are calculated using DESeq2 in the deseq2_qc.r script.
Software Versions
Software Versions lists versions of software tools extracted from file contents.
| Group | Software | Version |
|---|---|---|
| CUSTOM_GETCHROMSIZES | getchromsizes | 1.21 |
| CUSTOM_TX2GENE | python | 3.10.4 |
| DESEQ2_QC_STAR_SALMON | bioconductor-deseq2 | 1.46.0 |
| r-base | 4.4.2 | |
| DupRadar | bioconductor-dupradar | 1.32.0 |
| GTF2BED | perl | 5.26.2 |
| GTF_FILTER | python | 3.9.5 |
| MAKE_TRANSCRIPTS_FASTA | rsem | 1.3.1 |
| star | 2.7.10a | |
| PICARD_MARKDUPLICATES | picard | 3.1.1 |
| PRESEQ_LCEXTRAP | preseq | 3.2.0 |
| QUALIMAP_RNASEQ | qualimap | 2.3 |
| RSEQC_BAMSTAT | rseqc | 5.0.2 |
| RSEQC_INFEREXPERIMENT | rseqc | 5.0.2 |
| RSEQC_INNERDISTANCE | rseqc | 5.0.2 |
| RSEQC_JUNCTIONANNOTATION | rseqc | 5.0.2 |
| RSEQC_JUNCTIONSATURATION | rseqc | 5.0.2 |
| RSEQC_READDISTRIBUTION | rseqc | 5.0.2 |
| RSEQC_READDUPLICATION | rseqc | 5.0.2 |
| SALMON_QUANT | salmon | 1.10.3 |
| SAMTOOLS_FLAGSTAT | samtools | 1.21 |
| SAMTOOLS_IDXSTATS | samtools | 1.21 |
| SAMTOOLS_INDEX | samtools | 1.21 |
| SAMTOOLS_SORT_QUALIMAP | samtools | 1.21 |
| SAMTOOLS_STATS | samtools | 1.21 |
| SE_GENE_UNIFIED | bioconductor-summarizedexperiment | 1.32.0 |
| SE_TRANSCRIPT_UNIFIED | bioconductor-summarizedexperiment | 1.32.0 |
| SORTMERNA_INDEX | sortmerna | 4.3.7 |
| TXIMETA_TXIMPORT | bioconductor-tximeta | 1.20.1 |
| Workflow | Nextflow | 25.04.7 |
| nf-core/rnaseq | v3.22.2-g3816d48 |
nf-core/rnaseq Methods Description
Suggested text and references to use when describing pipeline usage within the methods section of a publication.https://github.com/nf-core/rnaseq
Methods
Data was processed using nf-core/rnaseq v3.22.2 (doi: 10.5281/zenodo.1400710) of the nf-core collection of workflows (Ewels et al., 2020), utilising reproducible software environments from the Bioconda (Grüning et al., 2018) and Biocontainers (da Veiga Leprevost et al., 2017) projects.
The pipeline was executed with Nextflow v25.04.7 (Di Tommaso et al., 2017) with the following command:
nextflow run nf-core/rnaseq -r 3.22.2 -c ../rnaseq.config -params-file ../params_rnaseq.json -resume -profile crg_slurm
References
- Di Tommaso, P., Chatzou, M., Floden, E. W., Barja, P. P., Palumbo, E., & Notredame, C. (2017). Nextflow enables reproducible computational workflows. Nature Biotechnology, 35(4), 316-319. doi: 10.1038/nbt.3820
- Ewels, P. A., Peltzer, A., Fillinger, S., Patel, H., Alneberg, J., Wilm, A., Garcia, M. U., Di Tommaso, P., & Nahnsen, S. (2020). The nf-core framework for community-curated bioinformatics pipelines. Nature Biotechnology, 38(3), 276-278. doi: 10.1038/s41587-020-0439-x
- Grüning, B., Dale, R., Sjödin, A., Chapman, B. A., Rowe, J., Tomkins-Tinch, C. H., Valieris, R., Köster, J., & Bioconda Team. (2018). Bioconda: sustainable and comprehensive software distribution for the life sciences. Nature Methods, 15(7), 475–476. doi: 10.1038/s41592-018-0046-7
- da Veiga Leprevost, F., Grüning, B. A., Alves Aflitos, S., Röst, H. L., Uszkoreit, J., Barsnes, H., Vaudel, M., Moreno, P., Gatto, L., Weber, J., Bai, M., Jimenez, R. C., Sachsenberg, T., Pfeuffer, J., Vera Alvarez, R., Griss, J., Nesvizhskii, A. I., & Perez-Riverol, Y. (2017). BioContainers: an open-source and community-driven framework for software standardization. Bioinformatics (Oxford, England), 33(16), 2580–2582. doi: 10.1093/bioinformatics/btx192
Notes:
- The command above does not include parameters contained in any configs or profiles that may have been used. Ensure the config file is also uploaded with your publication!
- You should also cite all software used within this run. Check the "Software Versions" of this report to get version information.
nf-core/rnaseq Workflow Summary
- this information is collected when the pipeline is started.https://github.com/nf-core/rnaseq
Input/output options
- input
- ../samplesheet_Drosophila.csv
- multiqc_title
- PTox-Production-phase2-Drosophila-include-dups
- outdir
- ./rnaseq_output
Reference genome options
- fasta
- ../../refs/Drosophila/genome.fa
- gtf
- ../../refs/Drosophila/genome_annotation.gtf
- gtf_extra_attributes
- gene
- star_index
- ../../rnaseq_run-Drosophila/rnaseq_output/genome/index/star
Read trimming options
- extra_fastp_args
- --length_required=20 --trim_poly_g --poly_g_min_len=20 --trim_poly_x --poly_x_min_len=15 --adapter_sequence=AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC --dont_eval_duplication --cut_tail
- trimmer
- fastp
Read filtering options
- remove_ribo_rna
- true
UMI options
- umi_discard_read
- 0
Alignment options
- extra_salmon_quant_args
- --noLengthCorrection
- extra_star_align_args
- --alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterMultimapNmax 20 --outFilterType BySJout --outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA
- stranded_threshold
- 0.7
Process skipping options
- skip_alignment
- true
- skip_bigwig
- true
- skip_biotype_qc
- true
- skip_preseq
- false
- skip_pseudo_alignment
- true
- skip_stringtie
- true
Generic options
- monochrome_logs
- true
- trace_report_suffix
- 2026-04-01_11-09-07
Core Nextflow options
- configFiles
- /users/rg/vntasis/.nextflow/assets/nf-core/rnaseq/nextflow.config, /users/project/precisiontox_008279_no_backup/production_phase2_data_dec2025/analyse_duplicated_data/rnaseq_run-Drosophila/../rnaseq.config
- containerEngine
- apptainer
- launchDir
- /users/project/precisiontox_008279_no_backup/production_phase2_data_dec2025/analyse_duplicated_data/rnaseq_run-Drosophila
- profile
- crg_slurm
- projectDir
- /users/rg/vntasis/.nextflow/assets/nf-core/rnaseq
- revision
- 3.22.2
- runName
- tiny_boyd
- userName
- vntasis
- workDir
- /users/project/precisiontox_008279_no_backup/production_phase2_data_dec2025/analyse_duplicated_data/rnaseq_run-Drosophila/work