DirectClean

Strand orientation, artifact removal, and chimeric read rescue for Oxford Nanopore direct-cDNA sequencing.

DirectClean processes raw ONT direct-cDNA FASTQ files and produces clean, oriented reads ready for transcript quantification and gene fusion analysis.

What it removes: foldback inversion reads (self-inverted artifacts) and reads that cannot be strand-oriented (missing primer signals).

What it rescues (chopped at the artifact junction, flanking sub-reads kept): reads containing internal TSO/RTP adapter junctions (concatemers from ligation) and reads containing homopolymer-mediated RT template switching junctions.

Performance on VCaP direct-cDNA data

Tested on 5.35M reads from the VCaP prostate cancer cell line:

Metric	Pychopper	DirectClean
Retention rate	57.6%	65.3%
FSM isoforms detected	17,873	20,535
Validated fusions detected (of 99)	37	49
Residual homopolymer artifacts	70,140	0

Why DirectClean?

Oxford Nanopore's Pychopper handles strand orientation and adapter-based read rescue, but direct-cDNA library preparation introduces additional artifact types that Pychopper does not address:

• Foldback inversions: the sequenced strand folds back on itself, producing a self-inverted chimeric read.
• Homopolymer-mediated RT template switching: during reverse transcription, the RT enzyme detaches at an A/T-rich region on one mRNA and re-primes on another, joining unrelated transcripts into a single chimeric read. These chimeras generate false gene fusion candidates and corrupt isoform quantification.

DirectClean integrates Breakinator and Restrander with novel detection and rescue algorithms into a single end-to-end pipeline.

Feature comparison

Capability	Pychopper	DirectClean
Strand orientation	✅	✅
Adapter concatemer rescue	✅ (requires terminal primers)	✅ (partial internal signal sufficient)
Foldback inversion removal	❌	✅
Homopolymer RT template switching detection	❌	✅
Rescue from unclassified reads	❌	✅

Pipeline architecture

Stage	Name	What it does
1	Breakinator	Remove foldback inversion artifacts
2	Restrander	Orient reads 5'→3', remove RTP-RTP / TSO-TSO artifacts, set aside unorientable reads
3	Unknowns Rescue	Recover orientable reads from Restrander unknowns via internal adapter detection and self-orientation
4	Adapter Rescue	Detect internal TSO/RTP adapters in oriented reads, chop and rescue sub-reads
5	Homopolymer Rescue	Detect RT template switching at A/T-rich chimeric junctions, chop and rescue sub-reads

Stages 1–2 remove definitively artifactual or unorientable reads. Stages 3, 4, and 5 never discard reads — they chop chimeric reads at artifact junctions and keep the flanking sub-reads as independent sequences.

How the homopolymer detector works

After minimap2 splice-aware alignment, DirectClean identifies chimeric reads (those with supplementary alignments mapping to different genomic loci). For each chimeric junction, a 10 bp sliding window scans the flanking sequence on both sides. A junction is flagged as an RT template switching artifact if any window satisfies both criteria:

• A/T base density ≥ 85%
• Longest consecutive A or T run ≥ 5 bp

Flagged reads are chopped at the artifact junction. Sub-reads ≥ 100 bp are written to the output; shorter fragments are discarded. Junctions on non-standard contigs (alt loci, unplaced scaffolds) are excluded via a standard-chromosome whitelist.

Installation

# Create environment with all dependencies
mamba env create -f environment.yml
mamba activate directclean

# Install DirectClean
poetry install

External tools (minimap2, samtools, breakinator, restrander) are included in the conda environment. To install them separately:

mamba install -c bioconda minimap2 samtools breakinator
mamba install -c genomedk restrander

Usage

directclean \
  -i raw_reads.fastq \
  -r genome.fa \
  -o results/ \
  -t 8 \
  -j gencode.v41.bed12

The -j flag provides a junction BED file for guided alignment (recommended: GENCODE annotation in BED12 format).

Key parameters

Flag	Default	Description
-i, --input	required	Raw FASTQ from ONT direct-cDNA sequencing
-r, --reference	required	Reference genome FASTA
-o, --output	required	Output directory
-t, --threads	4	Threads for minimap2, samtools, breakinator
-j, --junc-bed	none	Junction BED12 for guided alignment
--density-threshold	0.85	A/T density threshold for homopolymer detection
--min-run	5	Minimum consecutive A/T run length
--min-confidence	2	Minimum adapter signals (1–3) required to chop
--context-window	50	Bases flanking each junction for scanning
--html-report	off	Generate an interactive HTML summary report

Run directclean -h for the full list.

Output

results/
├── directclean.cleaned.fastq          All clean reads + rescued sub-reads
├── directclean.rescued.fastq          Sub-reads rescued by homopolymer chopping
├── directclean.homopolymer_report.tsv Per-read artifact classification
├── directclean.report.html            Interactive HTML report (if --html-report)
├── intermediates/
│   ├── directclean.no_foldback.fastq       After Stage 1
│   ├── directclean.restranded.fastq        After Stage 2
│   ├── directclean.unknowns_rescued.fastq  Stage 3 output
│   ├── directclean.rescued.fastq           After Stage 4
│   ├── directclean.merged.fastq            Stage 3 + Stage 4 merged
│   └── directclean.aligned.sorted.bam      Minimap2 alignment
└── reports/
    └── directclean.rescue_report.tsv  Stage 4 adapter rescue details

The primary output is directclean.cleaned.fastq. This file contains all reads that passed the pipeline plus rescued sub-reads from Stages 3, 4, and 5, ready for downstream transcript quantification (e.g., IsoQuant, FLAIR) and gene fusion calling (e.g., FusionSeeker, JAFFAL).

HTML Report

DirectClean generates an interactive HTML report with per-stage statistics and read flow visualization.

Citation

If you use DirectClean in your research, please cite our manuscript along with the foundational tools integrated into this pipeline:

• DirectClean: Guo, Q., Li, Y., & Yang, R. (2026). DirectClean: a comprehensive preprocessing toolkit for Oxford Nanopore direct-cDNA sequencing. Manuscript in preparation.
• Breakinator: Heinz, J. M., Meyerson, M., & Li, H. (2026). Detecting foldback artifacts in long-reads. BMC Genomics.
• Restrander: Schuster, J., Ritchie, M. E., & Gouil, Q. (2023). Restrander: rapid orientation and artefact removal for long-read cDNA data. NAR Genomics and Bioinformatics, 5(4), lqad108.

License

MIT

Contact

• Qingxiang Guo — qingxiang.guo@northwestern.edu
• Rendong Yang Lab — https://github.com/ylab-hi