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RustQC

Quick Start

This guide walks you through a basic RustQC analysis from start to finish.

Prerequisites

Section titled “Prerequisites”

The rustqc rna command runs all analyses in a single pass. It requires:

  • A duplicate-marked alignment file (BAM, SAM, or CRAM). Duplicates must be flagged with SAM flag 0x400 by a tool like Picard MarkDuplicates, samblaster, or sambamba.
  • Either a GTF annotation file (--gtf) or a BED12 gene model file (--bed). Both can be plain or gzip-compressed (.gz) — compression is detected automatically. With a GTF, all analyses run (dupRadar, featureCounts, and all 7 RSeQC tools). With a BED file, only the 7 RSeQC tools run. The two flags are mutually exclusive.

RNA-seq duplicate analysis

Section titled “RNA-seq duplicate analysis”

Run all RNA-seq QC analyses (dupRadar, featureCounts, and RSeQC tools) in a single pass:

rustqc rna sample.markdup.bam --gtf genes.gtf -p -o results/

This command:

  • Analyses sample.markdup.bam against genes.gtf
  • Uses paired-end mode (-p)
  • Writes all output to results/

Output

Section titled “Output”

Output files are organized into subdirectories by tool group. After running, you will find in the output directory:

FileDescription
dupradar/sample.markdup_dupMatrix.txtFull duplication matrix (TSV)
dupradar/sample.markdup_intercept_slope.txtFitted model parameters
dupradar/sample.markdup_duprateExpDens.pngDensity scatter plot
dupradar/sample.markdup_duprateExpBoxplot.pngDuplication rate boxplot
dupradar/sample.markdup_expressionHist.pngExpression histogram
featurecounts/sample.markdup.featureCounts.tsvGene-level read counts
featurecounts/sample.markdup.featureCounts.tsv.summaryCounting summary statistics
rseqc/read_duplication/sample.markdup.DupRate_plot.pngRead duplication rate plot
rseqc/junction_annotation/sample.markdup.splice_events.pngSplice events pie chart
rseqc/junction_annotation/sample.markdup.splice_junction.pngSplice junctions pie chart
rseqc/junction_saturation/sample.markdup.junctionSaturation_plot.pngJunction saturation plot
rseqc/inner_distance/sample.markdup.inner_distance_plot.pngInner distance histogram
rseqc/tin/sample.markdup.tin.xlsTranscript Integrity Numbers
samtools/sample.markdup.flagstatsamtools flagstat-compatible output
samtools/sample.markdup.idxstatssamtools idxstats-compatible output
samtools/sample.markdup.statssamtools stats SN-compatible output
preseq/sample.markdup.lc_extrap.txtLibrary complexity extrapolation
qualimap/rnaseq_qc_results.txtQualimap-compatible QC summary

Plus SVG versions of all plots, biotype count tables, and MultiQC-compatible report files. Use --flat-output to write all files directly to the output directory without subdirectories.

See dupRadar Outputs, featureCounts Outputs, RSeQC Outputs, Preseq Outputs, Samtools Outputs, and TIN & Gene Body Coverage for full details.

RSeQC quality control tools

Section titled “RSeQC quality control tools”

RustQC reimplements seven RSeQC tools, all integrated into the rustqc rna command. They run automatically alongside the dupRadar and featureCounts analyses:

# Run everything with a GTF: dupRadar + featureCounts + all 7 RSeQC tools
rustqc rna sample.markdup.bam --gtf genes.gtf -p -o results/


# Or run RSeQC tools only with a BED file (no dupRadar/featureCounts)
rustqc rna sample.markdup.bam --bed genes.bed -p -o results/

To disable specific RSeQC tools, use a YAML config file:

# config.yaml -- disable inner_distance
inner_distance:
  enabled: false
rustqc rna sample.markdup.bam --gtf genes.gtf -p -c config.yaml -o results/

See RSeQC Outputs for details on output files.

Multiple BAM files

Section titled “Multiple BAM files”

Multiple input files are accepted and processed sequentially, with each producing its own set of output files:

rustqc rna sample1.bam sample2.bam sample3.bam \
  --gtf genes.gtf -p -t 8 -o results/

The annotation file is parsed once and shared across all samples. Threads are distributed automatically among concurrent jobs.

Common options

Section titled “Common options”
# Stranded library (forward)
rustqc rna sample.bam --gtf genes.gtf -p -s 1


# Use 8 threads
rustqc rna sample.bam --gtf genes.gtf -p -t 8


# CRAM input with reference
rustqc rna sample.cram --gtf genes.gtf -p --reference genome.fa


# Gzip-compressed annotation files (auto-detected)
rustqc rna sample.bam --gtf genes.gtf.gz -p -o results/
rustqc rna sample.bam --bed genes.bed.gz -p -o results/


# Skip duplicate-marking validation
rustqc rna sample.bam --gtf genes.gtf -p --skip-dup-check


# Use a YAML config file
rustqc rna sample.bam --gtf genes.gtf -p --config config.yaml


# Custom MAPQ cutoff for RSeQC tools
rustqc rna sample.bam --gtf genes.gtf -p -q 20 -o results/

Next steps

Section titled “Next steps”