Differential analysis of gene regulation at transcript resolution with RNA-seq
Open accessAbstract
Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges. We present Cuffdiff 2, an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which we show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.
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Additional factors that interfere with intra-sample comparisons include changes in transcript length across samples or conditions, positional biases in coverage along the transcript, average fragment size, and the GC contents of genes.
“Additional factors that interfere with intra-sample comparisons include changes in transcript length across samples or conditions [50], positional biases in coverage along the transcript (which are accounted for in Cufflinks), average fragment size [43], and the GC contents of genes (corrected in the EDAseq package [21]).”
From A survey of best practices for RNA-seq data analysis (2016) · cites this paper as [50]
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“These results point to the need for new algorithm development to solve this difficult, but important, problem.”
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Fundamental Methods in Molecular Biology
Ijaz, S., & Hassan, S. (2026) · Molecular Biology Methods · doi:10.1007/978-981-92-2642-9_1
- Authors
- Trapnell, C., Hendrickson, D. G., Sauvageau, M., Goff, L., Rinn, J. L., & Pachter, L.
- Affiliations
- Harvard University (US)
- Broad Institute (US)
- University of California, Berkeley (US)
- Corresponding author
- Rinn, John L
- Pachter, Lior
- Venue
- Nature Biotechnology
- Published
- 2012-12-09
- Volume
- 31
- Issue
- 1
- Pages
- 46-53
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3869392
- OpenAlex
- W2166820820
- Cited by
- 3,492
- References
- 50
- Citations by year
- 2026: 76 · 2025: 135 · 2024: 184 · 2023: 177 · 2022: 229 · 2021: 309 · 2020: 346 · 2019: 339 · 2018: 372 · 2017: 373 · 2016: 357 · 2015: 283 · 2014: 224 · 2013: 84 · 2012: 2
- FWCI
- 76.42
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- CaltechAUTHORS (California Institute of Technology) (submittedVersion)
- Funders
- Damon Runyon Cancer Research Foundation
- National Institutes of Health (DP2OD006670)
- National Human Genome Research Institute (R01HG006129, P50HG006193)
- National Institute of General Medical Sciences (P01GM099117)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK094699)
- National Institute of Environmental Health Sciences (R01ES020260)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Keywords
- differential gene expression, differential splicing, isoform switching, HOXA1, lung fibroblasts, cell cycle progression, HeLa cells, measurement variability
- MeSH terms
- Homeobox A1 Protein, Fibroblasts, Gene Expression Regulation, HeLa Cells, Humans, Lung, RNA, Messenger, Transcription Factors, Sequence Analysis, RNA, Homeodomain Proteins
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