Ocean to Tree: Leveraging Single-Molecule RNA-Seq to Repair Genome Gene Models and Improve Phylogenomic Analysis of Gene and Species Evolution

Methods Mol Biol. 2024:2757:461-490. doi: 10.1007/978-1-0716-3642-8_19.


Understanding gene evolution across genomes and organisms, including ctenophores, can provide unexpected biological insights. It enables powerful integrative approaches that leverage sequence diversity to advance biomedicine. Sequencing and bioinformatic tools can be inexpensive and user-friendly, but numerous options and coding can intimidate new users. Distinct challenges exist in working with data from diverse species but may go unrecognized by researchers accustomed to gold-standard genomes. Here, we provide a high-level workflow and detailed pipeline to enable animal collection, single-molecule sequencing, and phylogenomic analysis of gene and species evolution. As a demonstration, we focus on (1) PacBio RNA-seq of the genome-sequenced ctenophore Mnemiopsis leidyi, (2) diversity and evolution of the mechanosensitive ion channel Piezo in genetic models and basal-branching animals, and (3) associated challenges and solutions to working with diverse species and genomes, including gene model updating and repair using single-molecule RNA-seq. We provide a Python Jupyter Notebook version of our pipeline (GitHub Repository: Ctenophore-Ocean-To-Tree-2023 https://github.com/000generic/Ctenophore-Ocean-To-Tree-2023 ) that can be run for free in the Google Colab cloud to replicate our findings or modified for specific or greater use. Our protocol enables users to design new sequencing projects in ctenophores, marine invertebrates, or other novel organisms. It provides a simple, comprehensive platform that can ease new user entry into running their evolutionary sequence analyses.

Keywords: Ctenophora; De novo transcriptome; Gene family tree; Mechanosensitive ion channels; Mnemiopsis; Phylogenetic trees; Phylogenomics; Piezo; Single-molecule sequencing PacBio SMRT.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Computational Biology / methods
  • Ctenophora* / classification
  • Ctenophora* / genetics
  • Evolution, Molecular*
  • Genome / genetics
  • Genomics / methods
  • Models, Genetic
  • Phylogeny*
  • RNA-Seq* / methods
  • Software