A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

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 purification kit (Table of Materials)
      NOTE: There are protocols and kits that may work better for different species and tissu....

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 datasets^4,34,35. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bioanalyzer-DNA kit	Agilent	5067-4626	wet lab materials
Bioanalyzer-RNA kit	Agilent	5067-1513	wet lab materials
BLAST+ v. 2.8.1			On 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. 1.57.0			On computer cluster
Bowtie v. 1.0.0			On 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. 2.2.1			On computer cluster
edgeR v. 3.26.8 (in R)			In Rstudio https://bioconductor.org/packages/release/bioc/html/edgeR.html
gcc v. 6.4.0			On computer cluster
Java v. 11.0.2			On computer cluster
MEGA7 (on your PC)			On local computer https://www.megasoftware.net
MEGAX v. 0.1			On local computer https://www.megasoftware.net
NucleoSpin RNA II kit	Macherey-Nagel	740955.5	wet lab materials
perl 5.30.3			On computer cluster
python			On computer cluster
Qubit 2.0 Fluorometer	ThermoFisher	Q32866	wet lab materials
R v.4.0.0			On computer cluster https://cran.r-project.org/src/base/R-4/
RNAlater	ThermoFisher	AM7021	wet lab materials
RNeasy kit	Qiagen	74104	wet lab materials
RSEM v. 1.3.0			Computer software https://deweylab.github.io/RSEM/
RStudio v. 1.2.1335			On local computer https://rstudio.com/products/rstudio/download/#download
Samtools v. 1.3			Computer software
SRA Toolkit v. 2.8.1			On computer cluster https://github.com/ncbi/sra-tools/wiki/01.-Downloading-SRA-Toolkit
STAR v. 2.6.0c			On computer cluster https://github.com/alexdobin/STAR
StringTie v. 1.3.4d			On computer cluster https://ccb.jhu.edu/software/stringtie/
Transdecoder v. 5.5.0			On computer cluster https://github.com/TransDecoder/TransDecoder/releases
Trimmomatic v. 0.35			On computer cluster http://www.usadellab.org/cms/?page=trimmomatic
Trinity v.2.8.5			On computer cluster https://github.com/trinityrnaseq/trinityrnaseq/releases
TRIzol	ThermoFisher	15596018	wet lab materials
TruSeq RNA Library Prep Kit v2	Illumina	RS-122-2001	wet lab materials
TURBO DNA-free Kit	ThermoFisher	AM1907	wet lab materials
*Downloads and installation on the computer cluster may require root access. Contact your network administrator.