ArticleRNA (New York, N.Y.)2025
Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing.
Article in RNA (New York, N.Y.), 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed.
- Review
- qMAP decodes RNA fragmentation dynamics in development and disease.Molecular systems biology · 2026Article
- OTTR-CLASH: improved biochemical and bioinformatic identification of Argonaute 2-mediated microRNA-target RNA interactions.bioRxiv : the preprint server for biology · 2026Article
- The regulation, function and disease relevance of cytoplasmic tRNAs.Nature reviews. Molecular cell biology · 2026Review
- Metabolic stress reveals widespread accumulation of cap-unmethylated RNAs.bioRxiv : the preprint server for biology · 2026Article
- Small RNA and Toll-like receptor interactions: origins and disease mechanisms.Trends in biochemical sciences · 2025Review
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6 authors.
Funding
Abstract
Sequencing RNAs that are biologically processed or degraded to less than ∼100 nt typically involves multistep, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced ordered two-template relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated
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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.