PUBLICATION
Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing
- Authors
- Begik, O., Diensthuber, G., Liu, H., Delgado-Tejedor, A., Kontur, C., Niazi, A.M., Valen, E., Giraldez, A.J., Beaudoin, J.D., Mattick, J.S., Novoa, E.M.
- ID
- ZDB-PUB-221220-16
- Date
- 2022
- Source
- Nature Methods 20(1): 75-85 (Journal)
- Registered Authors
- Giraldez, Antonio, Kontur, Cassandra
- Keywords
- none
- MeSH Terms
-
- Animals
- DNA, Complementary/genetics
- Gene Expression Profiling
- Mice
- Nanopores*
- Poly A/genetics
- Poly A/metabolism
- RNA/genetics
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Sequence Analysis, RNA/methods
- Transcriptome*
- Zebrafish/genetics
- Zebrafish/metabolism
- PubMed
- 36536091 Full text @ Nat. Methods
Citation
Begik, O., Diensthuber, G., Liu, H., Delgado-Tejedor, A., Kontur, C., Niazi, A.M., Valen, E., Giraldez, A.J., Beaudoin, J.D., Mattick, J.S., Novoa, E.M. (2022) Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nature Methods. 20(1):75-85.
Abstract
RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping