DirectClean: artifact-aware preprocessing and read rescue for Oxford Nanopore direct-cDNA transcriptome sequencing
- 1Department of Urology, Northwestern University Feinberg School of Medicine , 303 E Superior St, Chicago, IL 60611 ,
- 2Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine , 303 E Superior St, Chicago, IL 60611 ,
Abstract
Oxford Nanopore direct-cDNA sequencing enables full-length transcriptome profiling without PCR amplification, but recurrent chimeric artifacts introduced during reverse transcription and library preparation can compromise isoform reconstruction and gene-fusion analysis. These include foldback inversions, aberrant primer configurations, internal adapter concatemers, and homopolymer-mediated template-switching events. Existing preprocessing strategies primarily address primer-based orientation and adapter detection, leaving several direct-cDNA-specific artifact classes unresolved and often discarding reads that still contain usable transcript sequence. We developed DirectClean, an artifact-aware preprocessing and read-rescue framework integrating strand orientation, artifact removal, and junction-level splitting of recoverable chimeric reads. Benchmarking across four direct-cDNA datasets showed consistently higher read and base retention with DirectClean than with Pychopper. In VCaP, DirectClean retained 65.2% of reads compared with 57.6% for Pychopper, supported additional transcripts, and reduced the chimeric read rate from 30.9% in raw data to 16.9%. Semi-synthetic validation demonstrated accurate rescue of adapter-associated and A/T-rich chimeric reads, and matched real-data analyses showed no enrichment of novel transcript models in split-derived reads relative to matched untouched reads. DirectClean provides a reproducible, openly available framework for artifact-aware preprocessing of direct-cDNA transcriptomes and improves the reliability of downstream isoform and fusion analyses.
Related
- Characterization of METTL3/14-mediated m⁶A modification in human transcriptome using Nanopore direct RNA sequencing
- Integrative Nanopore and Illumina sequencing reveals age-associated tRNA modification and CCA-tail dynamics in yeast
- NANOCUTSIGHT: A NANOPORE-SEQUENCING APPROACH AND ANALYSIS PIPELINE TO ASSESS GENOME EDITING EFFICACY IN VARIOUS CELL POPULATIONS
- Nanopore sequencing identifies a new Tyr::CreERT2 allele circulating in existing mouse stocks
- Nanopore sequencing reveals the DNA replication timing programme of the malaria parasite Plasmodium falciparum
BibTeX
@article{guo2026directclean,
title = {{DirectClean: artifact-aware preprocessing and read rescue for Oxford Nanopore direct-cDNA transcriptome sequencing}},
author = {Qingxiang Guo and Yangyang Li and Rendong Yang},
journal = {NAR Genomics and Bioinformatics},
year = {2026},
month = oct,
doi = {10.1093/nargab/lqag132},
url = {https://academic.oup.com/nargab/article/doi/10.1093/nargab/lqag132/9005802},
note = {CC BY},
}
RIS
TY - JOUR
TI - DirectClean: artifact-aware preprocessing and read rescue for Oxford Nanopore direct-cDNA transcriptome sequencing
AU - Qingxiang Guo
AU - Yangyang Li
AU - Rendong Yang
PY - 2026
DA - 2026/10/31
JO - NAR Genomics and Bioinformatics
DO - 10.1093/nargab/lqag132
UR - https://academic.oup.com/nargab/article/doi/10.1093/nargab/lqag132/9005802
AB - Oxford Nanopore direct-cDNA sequencing enables full-length transcriptome profiling without PCR amplification, but recurrent chimeric artifacts introduced during reverse transcription and library preparation can compromise isoform reconstruction and gene-fusion analysis. These include foldback inversions, aberrant primer configurations, internal adapter concatemers, and homopolymer-mediated template-switching events. Existing preprocessing strategies primarily address primer-based orientation and adapter detection, leaving several direct-cDNA-specific artifact classes unresolved and often discarding reads that still contain usable transcript sequence. We developed DirectClean, an artifact-aware preprocessing and read-rescue framework integrating strand orientation, artifact removal, and junction-level splitting of recoverable chimeric reads. Benchmarking across four direct-cDNA datasets showed consistently higher read and base retention with DirectClean than with Pychopper. In VCaP, DirectClean retained 65.2% of reads compared with 57.6% for Pychopper, supported additional transcripts, and reduced the chimeric read rate from 30.9% in raw data to 16.9%. Semi-synthetic validation demonstrated accurate rescue of adapter-associated and A/T-rich chimeric reads, and matched real-data analyses showed no enrichment of novel transcript models in split-derived reads relative to matched untouched reads. DirectClean provides a reproducible, openly available framework for artifact-aware preprocessing of direct-cDNA transcriptomes and improves the reliability of downstream isoform and fusion analyses.
SN - 2631-9268
ER -