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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

Abstract

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

Introduction

Advances in sequencing technologies have facilitated the sequencing of genomes and transcriptomes of non-model organisms. In addition to the increased feasibility of sequencing DNA and RNA from many organisms, an abundance of data is publicly available to study genes of interest. The purpose of this protocol is to provide bioinformatic steps to investigate the molecular evolution and expression of genes that may play an important role in the organism of interest.

Investigating the evolution of a gene or gene family can provide insight into the evolution of biological systems. Members of a gene family are typically determined by identifying ....

Protocol

This protocol follows UC Irvine animal care guidelines.

1. RNA-seq library preparation

  1. Isolate RNA using the following methods.
    1. Collect samples. If RNA is to be extracted at a later time, flash freeze the sample or place in RNA storage solution15 (Table of Materials).
    2. Euthanize and dissect the organism to separate tissues of interest.
    3. Extract total RNA using an extraction kit and purify the RNA using an RNA .......

Representative Results

The methods above are summarized in Figure 1 and were applied to a data set of Hydra vulgaris tissues. H. vulgaris is a fresh-water invertebrate that belongs to the phylum Cnidaria which also includes corals, jellyfish, and sea anemones. H. vulgaris can reproduce asexually by budding and they can regenerate their head and foot when bisected. In this study, we aimed to investigate the evolution and expression of opsin genes in Hydra

Discussion

The purpose of this protocol is to provide an outline of the steps for characterizing a gene family using RNA-seq data. These methods have been proven to work for a variety of species and datasets4,34,35. The pipeline established here has been simplified and should be easy enough to be followed by a novice in bioinformatics. The significance of the protocol is that it outlines all the steps and necessary programs to complete a p.......

Acknowledgements

We thank Adriana Briscoe, Gil Smith, Rabi Murad and Aline G. Rangel for advice and guidance in incorporating some of these steps into our workflow. We are also grateful to Katherine Williams, Elisabeth Rebboah, and Natasha Picciani for comments on the manuscript. This work was supported in part by a George E. Hewitt Foundation for Medical research fellowship to A.M.M.

....

Materials

NameCompanyCatalog NumberComments
Bioanalyzer-DNA kitAgilent5067-4626wet lab materials
Bioanalyzer-RNA kitAgilent5067-1513wet lab materials
BLAST+ v. 2.8.1On computer cluster*
https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
Blast2GO (on your PC)On local computer
https://www.blast2go.com/b2g-register-basic
boost v. 1.57.0On computer cluster
Bowtie v. 1.0.0On computer cluster
https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.3.0/
Computing cluster (highly recommended)NOTE: Analyses of genomic data are best done on a high-performance computing cluster because files are very large.
Cufflinks v. 2.2.1On computer cluster
edgeR v. 3.26.8 (in R)In Rstudio
https://bioconductor.org/packages/release/bioc/html/edgeR.html
gcc v. 6.4.0On computer cluster
Java v. 11.0.2On computer cluster
MEGA7 (on your PC)On local computer
https://www.megasoftware.net
MEGAX v. 0.1On local computer
https://www.megasoftware.net
NucleoSpin RNA II kitMacherey-Nagel740955.5wet lab materials
perl 5.30.3On computer cluster
pythonOn computer cluster
Qubit 2.0 FluorometerThermoFisherQ32866wet lab materials
R v.4.0.0On computer cluster
https://cran.r-project.org/src/base/R-4/
RNAlaterThermoFisherAM7021wet lab materials
RNeasy kitQiagen74104wet lab materials
RSEM v. 1.3.0Computer software
https://deweylab.github.io/RSEM/
RStudio v. 1.2.1335On local computer
https://rstudio.com/products/rstudio/download/#download
Samtools v. 1.3Computer software
SRA Toolkit v. 2.8.1On computer cluster
https://github.com/ncbi/sra-tools/wiki/01.-Downloading-SRA-Toolkit
STAR v. 2.6.0cOn computer cluster
https://github.com/alexdobin/STAR
StringTie v. 1.3.4dOn computer cluster
https://ccb.jhu.edu/software/stringtie/
Transdecoder v. 5.5.0On computer cluster
https://github.com/TransDecoder/TransDecoder/releases
Trimmomatic v. 0.35On computer cluster
http://www.usadellab.org/cms/?page=trimmomatic
Trinity v.2.8.5On computer cluster
https://github.com/trinityrnaseq/trinityrnaseq/releases
TRIzolThermoFisher15596018wet lab materials
TruSeq RNA Library Prep Kit v2IlluminaRS-122-2001wet lab materials
TURBO DNA-free KitThermoFisherAM1907wet lab materials
*Downloads and installation on the computer cluster may require root access. Contact your network administrator.

References

  1. Lespinet, O., Wolf, Y. I., Koonin, E. V., Aravind, L. The role of lineage-specific gene family expansion in the evolution of eukaryotes. Genome Research. 12 (7), 1048-1059 (2002).
  2. Gabaldón, T., Koonin, E. V.

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Bioinformatics PipelineMolecular EvolutionGene ExpressionRNA seqSRA ToolkitNCBIFASTQReference GenomeGTFFASTAGene MappingGene ExpressionBLASTHomologous GenesGene AnnotationMEGA

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