A Bioinformatics Pipeline to

Explore Transcriptional Regulation

Henrique Moura Dias

Marie-Anne Van Sluys

Department of Botany - Institute of Bioscience

University of São Paulo

September, 2019
 OVERVIEW AND OUTLINE
Part 1: Background and theory
• Galaxy
• A brief review of gene regulation
• Differential expression analysis
• Transcriptomic data: Microarrays and RNA-seq*
• Database
• Analysis:
• Quality control, preprocessing
• Statistical methods for differential expression analysis
• Functional analysis of differentially-expressed genes

Part 2: Hands-on activities to explore differentially-expressed genes

• Activity: RNA-Seq data: Genome-wide regulation
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Part 1: Background and theory

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META TOOLS FROM ANALYSIS

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A BRIEF REVIEW OF GENE REGULATION

What is transcriptome?

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GENE REGULATION OCCURS AT MANY LEVELS FROM
DNA → mRNA → PROTEIN

Image: ArneLH

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INTRODUCTION TO GENE REGULATION AND TRANSCRIPTION

Example: Melanin production in skin is a

transcriptional response to UV light

Images : Daniel Horspool; Scientific Animations.com.

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WHAT INFORMATION CAN BE OBTAINED FROM THE
TRANSCRIPTOME ANALYSIS?

Gene identification and molecular pathways involved in biological process

Identify molecular marks for diagnostic disease

Provide clues about function genes no still characterized

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DIFFERENTIAL EXPRESSION ANALYSIS: TOOLS AND TECHNIQUES, OUTCOMES

Microarray

Data collection

Quality control and preprocessing Gene Network

Differential expression analysis
Expression and functional clustering
RNA analysis Network analysis
using microarray
or RNA-Seq

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DIFFERENCES BETWEEN MICROARRAY AND RNA-SEQ

Microarray RNA-Seq
• Explore changes in expression of • Explore expression changes in known
known transcripts transcripts and find novel ones
• Dynamic range: 44-fold • Dynamic range: 9,560-fold
• Saturation and loss of signal • Highly expressed genes may
• Simple data analysis consume majority of reads
approximately < 1 GB • Complex data analysis
approximately > 5 GB

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OBTAINING DATA FOR TRANSCRIPTOMIC ANALYSIS: MICROARRAYS
AND RNA-SEQUENCING

Isolate RNA from samples

Examples:
• Leaf versus root
• Wild-type versus mutant
• Condition 1 vs Condition 2
Reverse transcribe
to produce cDNA

Quantify: Microarray
or RNA-Seq

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SHORT-READ, LONG-READ
AND DIRECT RNAseq
TECHNOLOGIES AND
WORKFLOW

Short-read (> 1000 bp)

Long-read (> 500 bp)

Adapted from Stark, R. ; Grzelak, M. and Hadfaptedield, J. (2019). RNA sequencing: the teenage years. Nature. DOI: s41576-019-0150-2.

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RNA-SEQUENCING: QUANTIFICATION BY SEQUENCING

Mutant
Wild type

Adapted from Griffith, M., Walker, J.R., Spies, N.C., Ainscough, B.J. and Griffith, O.L. (2015). Informatics for RNA Sequencing: A Web Resource for Analysis on the Cloud. PLOS Comp. Biol. 11: e1004393.

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OVERVIEW AND OUTLINE

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OVERVIEW AND OUTLINE

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RNAseq DATA ANALYSIS WORKFLOW FOR DIFFERENTIAL GENE EXPRESSION

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Part 2: Hands-on activities to explore differentially-expressed genes

Activity: RNA-Seq data: Genome-wide regulation

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 Thanks!

diash@usp.br

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