ArticleBMC genomics2022
Poly(a) selection introduces bias and undue noise in direct RNA-sequencing.
Article in BMC genomics, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed, 39 citations in OpenAlex.
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- Next-Generation Sequencing: A Review of Its Transformative Impact on Cancer Diagnosis, Treatment, and Resistance Management.Diagnostics (Basel, Switzerland) · 2025Review
- Nanopore direct RNA sequencing of human transcriptomes reveals the complexity of mRNA modifications and crosstalk between regulatory features.Cell genomics · 2025Article
- Endonucleolytic cleavage is the primary mechanism of decay elicited byGenome research · 2025Article
- Long-read RNA sequencing reveals allele-specificGenome research · 2025Article
- Detection of mRNA Transcript Variants.Genes · 2025Review
- Single-Cell RNA Sequencing Reveals Extensive Heterogeneity and Unique Gene Trajectories in Non-Transformed and Transformed Human Lung Epithelial Cells: Insights into the Role of LncRNAs in Tumor Heterogeneity.International journal of molecular sciences · 2025Article
- RiboTag RNA Sequencing Identifies Local Translation of HSP70 in Astrocyte Endfeet After Cerebral Ischemia.International journal of molecular sciences · 2025Article
- Isolation of a Specific Transcript Using Biotinylated Antisense Probes.Methods in molecular biology (Clifton, N.J.) · 2025Article
- TAILcaller: an R package for analyzing differences in poly(A) tail length for Oxford Nanopore RNA sequencing.Bioinformatics advances · 2025Article
- Latitude- and depth-driven divergence in protist trophic strategies revealed by a machine learning model.Frontiers in microbiology · 2025Article
- Insights into the human cDNA: A descriptive study using library screening in yeast.Journal, genetic engineering & biotechnology · 2024Article
- Long-read RNA sequencing of archival tissues reveals novel genes and transcripts associated with clear cell renal cell carcinoma recurrence and immune evasion.Genome research · 2024Article
- High-resolution reconstruction of aRNA (New York, N.Y.) · 2024Article
- Long-read RNA sequencing reveals allele-specific NbioRxiv : the preprint server for biology · 2024Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
backgroundGenome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.
resultsHere, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.
conclusionsOur work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.