Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Summary

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Abstract

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Introduction

The first sequencing of the human genome, performed using Fred Sanger's dideoxynucleotide-sequencing method, took 10 years, and cost an estimated US $3 billion^1,2. However, in a little over a decade since its inception, Next-Generation Sequencing (NGS) technology has made it possible to sequence the entire human genome within two weeks and for US $1,000. New NGS instruments that allow ever-increasing speeds of sequencing-data collection with incredible efficiency, along with sharp reductions in cost, are revolutionizing modern biology in unimaginable ways as genome sequencing projects are rapidly becoming comm....

Protocol

1. RNA Isolation

1. Precautionary measures
   1. Wipe down the entire working surface, instruments and pipettes using a commercially-available RNase spray to eliminate any RNases present.
   2. Wear gloves at all times, regularly changing them with fresh ones during the different steps of the protocol.
   3. Use only filter tips and keep all samples on ice as much as possible to avoid RNA degradation.
      NOTE: In order to obtain the best data from NGS platforms, it is critical to begin with high-quality RNA. RNA isolation and preparation methods vary depending on sample origin, method of sequencing and investigator preferen....

Results

In C. elegans, elimination of the germline stem cells (GSCs) extends lifespan, enhances stress resilience, and elevates body fat^24,28. Loss of GSCs, either brought about by laser-ablation or by mutations such as glp-1, causes lifespan extension through activation of a network of transcription factors²⁹. One such factor, TCER-1, encodes the worm homolog of the human transcription elongation.......

Discussion

Significance of the Galaxy Sequencing Platform in Modern Biology

The Galaxy Project has become instrumental in helping biologists without bioinformatics training to process and analyze high-throughput sequencing data in a fast and efficient manner. Once considered a herculean task, this publicly-available platform has made running complex bioinformatics algorithms to analyze NGS data a straightforward, reliable, and easy process. Apart from hosting a wide range of bioinformatics tools, the key to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNase spray	Fisher Scientific	21-402-178
Trizol	Ambion	15596026
Sonicator	Sonics Vibra Cell	VCX130
Centrifuge	Eppendorf	5415C
chloroform	Sigma Aldrich	288306
2-propanol	Fisher Scientific	A416P-4
Ethanol	Decon Labs	2705HC
RNase-free water	Fisher Scientific	BP561-1
Bioanalyzer	Agilent	G2940CA
Mac/PC