ArticleProceedings of the National Academy of Sciences of the United States of America2012
Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 149 papers.
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Who cites it
149 citing papers in PubMed.
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- KLF2 expression in IgG plasma cells at their induction site regulates the migration program.The Journal of experimental medicine · 2025Article
- Global identification and regulatory network analysis reveal the significant roles of lncRNAs during anther and pollen development in Arabidopsis.Plant cell reports · 2025Article
- Finding differentially expressed genes between cell fates predicted by image-based deep learning.Biophysics and physicobiology · 2025Article
- Transcriptome and Temporal Transcriptome Analyses in Single Cells.International journal of molecular sciences · 2024Review
- Long journey of 16S rRNA-amplicon sequencing toward cell-based functional bacterial microbiota characterization.iMetaOmics · 2024Review
- Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile.eLife · 2024Article
- Differential selection of yield and quality traits has shaped genomic signatures of cowpea domestication and improvement.Nature genetics · 2024Article
- Current advancements in B-cell receptor sequencing fast-track the development of synthetic antibodies.Molecular biology reports · 2024Review
- Article
- Methodologies for bacterial ribonuclease characterization using RNA-seq.FEMS microbiology reviews · 2023Review
- CD45 alleviates airway inflammation and lung fibrosis by limiting expansion and activation of ILC2s.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- A critical spotlight on the paradigms of FFPE-DNA sequencing.Nucleic acids research · 2023Review
- Sulforaphane Inhibits Exhaustive Exercise-Induced Liver Injury and Transcriptome-Based Mechanism Analysis.Nutrients · 2023Article
89 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors.
Funding
Abstract
RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq.
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