Introductory RNA-seq Transcriptome Profiling

Before we start: Align sequence reads to the reference genomeThe most time-consuming part of the analysis is doing the alignments of the reads (in Sanger fastq format) for all replicates against the reference genome.

Overview: This training module is designed to provide a hands on experience in using RNA-Seq for transcriptome profiling.

Question: How well is the annotated transcriptome represented in RNA-seq data in Arabidopsis WT and hy5 genetic backgrounds?

How can we compare gene expression levels in the two samples?

RNA-seq in the Discovery Environment

Scientific Objective

LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF).

Mutations in the HY5 gene cause aberrant phenotypes in Arabidopsis morphology, pigmentation and hormonal response.

We will use RNA-seq to compare the transcriptomes of seedlings from WT and hy5 genetic backgrounds to identify HY5-regulated genes.

Samples

• Experimental data downloaded from the NCBI Short Read Archive (GEO:GSM613465 and GEO:GSM613466)

• Two replicates each of RNA-seq runs for Wild-type and hy5 mutant seedlings.

Specific Objectives

By the end of this module, you should

1)Be more familiar with the DE user interface

2)Understand the starting data for RNA-seq analysis

3)Be able to align short sequence reads with a reference genome in the DE

4)Be able to analyze differential gene expression in the DE

5)Be able to use DE text manipulation tools to explore the gene expression data

RNA-Seq Conceptual Overview

Image source: http://www.bgisequence.com

@SRR070570.4 HWUSI-EAS455:3:1:1:1096 length=41CAAGGCCCGGGAACGAATTCACCGCCGTATGGCTGACCGGC+BA?39AAA933BA05>A@A=?4,9#################@SRR070570.12 HWUSI-EAS455:3:1:2:1592 length=41GAGGCGTTGACGGGAAAAGGGATATTAGCTCAGCTGAATCT+@=:9>5+.5=?@<6>A?@6+2?:</7>,%1/=0/7/>48##@SRR070570.13 HWUSI-EAS455:3:1:2:869 length=41TGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCA+A;BAA6=A3=ABBBA84B<&78A@BA=(@B>AB2@>B@/9?@SRR070570.32 HWUSI-EAS455:3:1:4:1075 length=41CAGTAGTTGAGCTCCATGCGAAATAGACTAGTTGGTACCAC+BB9?A@>AABBBB@BCA?A8BBBAB4B@BC71=?9;B:3B?@SRR070570.40 HWUSI-EAS455:3:1:5:238 length=41AAAAGGGTAAAAGCTCGTTTGATTCTTATTTTCAGTACGAA+BBB?06-8BB@B17>9)=A91?>>8>*@<A<>>@1:B>(B@@SRR070570.44 HWUSI-EAS455:3:1:5:1871 length=41GTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTGTAAG+BBBCBCCBBBBBA@BBCCB+ABBCB@B@BB@:BAA@B@BB>@SRR070570.46 HWUSI-EAS455:3:1:5:1981 length=41GAACAACAAAACCTATCCTTAACGGGATGGTACTCACTTTC+?A>-?B;BCBBB@BC@/>A<BB:?<?B?=75?:9@@@3=>:

Bioinformagician

$ tophat -p 8 -G genes.gtf -o C1_R1_thout genome C1_R1_1.fq C1_R1_2.fq$ tophat -p 8 -G genes.gtf -o C1_R2_thout genome C1_R2_1.fq C1_R2_2.fq$ tophat -p 8 -G genes.gtf -o C1_R3_thout genome C1_R3_1.fq C1_R3_2.fq$ tophat -p 8 -G genes.gtf -o C2_R1_thout genome C2_R1_1.fq C1_R1_2.fq$ tophat -p 8 -G genes.gtf -o C2_R2_thout genome C2_R2_1.fq C1_R2_2.fq$ tophat -p 8 -G genes.gtf -o C2_R3_thout genome C2_R3_1.fq C1_R3_2.fq

$ cufflinks -p 8 -o C1_R1_clout C1_R1_thout/accepted_hits.bam$ cufflinks -p 8 -o C1_R2_clout C1_R2_thout/accepted_hits.bam$ cufflinks -p 8 -o C1_R3_clout C1_R3_thout/accepted_hits.bam$ cufflinks -p 8 -o C2_R1_clout C2_R1_thout/accepted_hits.bam$ cufflinks -p 8 -o C2_R2_clout C2_R2_thout/accepted_hits.bam$ cufflinks -p 8 -o C2_R3_clout C2_R3_thout/accepted_hits.bam

$ cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt

$ cuffdiff -o diff_out -b genome.fa -p 8 –L C1,C2 -u merged_asm/merged.gtf \./C1_R1_thout/accepted_hits.bam,./C1_R2_thout/accepted_hits.bam,\./C1_R3_thout/accepted_hits.bam \./C2_R1_thout/accepted_hits.bam,\./C2_R3_thout/accepted_hits.bam,./C2_R2_thout/accepted_hits.bam

Your RNA-Seq Data

Your transformed RNA-Seq Data

RNA-Seq Analysis Workflow

Tophat (bowtie)

Cufflinks

Cuffmerge

Cuffdiff

CummeRbund

Your Data

iPlant Data Store

FASTQ

Disco

very E

nviro

nm

en

t Atm

osphe

re

Quick Summary

Find D

iffere

ntially

Expre

ssed genes

Align to

Genom

e: TopHat

View Alig

nments

: IGV

Differe

ntial E

xpressio

n: CuffD

iff

Download R

eads from

SRA

Export Reads to

FASTQ

Import SRA data from NCBI SRA

Extract FASTQ files from the

downloaded SRA archives

Pre-Configured: Getting the RNA-seq Data

Examining Data Quality with fastQC

Examining Data Quality with fastQC

RNA-Seq Workflow Overview

Align the four FASTQ files to Arabidopsis genome using TopHat

Step 1: Align Reads to the Genome

Built-in ref. genomes

User provided ref. genomes

A single FASTQ file

Folder with >= 1 FASTQ files

TopHat

• TopHat is one of many applications for aligning short sequence reads to a reference genome.

• It uses the BOWTIE aligner internally.

• Other alternatives are BWA, MAQ, OLego, Stampy, Novoalign, etc.

RNA-seq Sample Read Statistics

• Genome alignments from TopHat were saved as BAM files, the binary version of SAM (samtools.sourceforge.net/).

• Reads retained by TopHat are shown below

Sequence run WT-1 WT-2 hy5-1 hy5-2

Reads 10,866,702 10,276,268 13,410,011 12,471,462

Seq. (Mbase) 445.5 421.3 549.8 511.3

Index BAM files using SAMtools

Prepare BAM files for viewing

Using IGV in Atmosphere

1. We already Launched an instance of NGS Viewers in Atmosphere

2. Use VNClient to connect to your remote desktop

Pre-configured VM for NGS Viewers

The Integrative Genomics Viewer (IGV) is a high-performance visualization tool for interactive exploration of large, integrated genomic datasets. It supports a wide variety of data types, including array-based and next-generation sequence data, and genomic annotations.

Use IGV to inspect outputs from TopHat

http://www.broadinstitute.org/igv/

Integrated Genomics Viewer (IGV)

ATG44120 (12S seed storage protein) significantly down-regulated in hy5 mutantBackground (> 9-fold p=0). Compare to gene on right lacking differential expression

RNA-Seq Workflow Overview

CuffDiff

• CuffLinks is a program that assembles aligned RNA-Seq reads into transcripts, estimates their abundances, and tests for differential expression and regulation transcriptome-wide.

• CuffDiff is a program within CuffLinks that compares transcript abundance between samples

Examining Differential Gene Expression

Examining the Gene Expression Data

Filter CuffDiff results for up or down-regulated gene expression in hy5 seedlings

Differentially expressed genes

Differentially expressed genes

Example filtered CuffDiff results generated with the Filter_CuffDiff_Results to1)Select genes with minimum two-fold expression difference2)Select genes with significant differential expression (q <= 0.05)3)Add gene descriptions

Coming Soon: Downstream Analysis with cummeRbund

Coming Soon: Support for Paired End Reads and Other Sequencing Platforms

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