An Introduction to RNA-Seq Transcriptome Profiling with iPlant

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 demonstrate a workflow in the iPlant Discovery Environment using RNA-Seq for transcriptome profiling.

Question: How can we compare gene expression levels using RNA-Seq data in Arabidopsis WT and hy5 genetic backgrounds?

RNA-seq in the Discovery Environment

Scientific Objective

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

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

We will use RNA-seq to compare WT and hy5 to identify HY5-regulated genes.

Source: http://www.gla.ac.uk/media/media_73736_en.jpg

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.

RNA-Seq Conceptual Overview

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

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

RNA-Seq Data

@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=>:

…Now What?

@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=>:

Bioinformatician

0100110

10 1

The Tuxedo Protocol

$ 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

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The iPlant Discovery Environment

The iPlant Discovery Environment

The iPlant Discovery Environment

The iPlant Discovery Environment

Import SRA data from NCBI SRA

Extract FASTQ files from the

downloaded SRA archives

Getting the RNA-Seq Data

Staged Data

Examining Data Quality with fastQC

Tophat

Tophat in the Discovery Environment

Align the four FASTQ files to Arabidopsis genome using Tophat

Align Reads to the Genome

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.

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

Assembling the Transcripts

Cufflinks in the Discovery Environment

Cufflinks

Merging the Transcriptomes

Cufffmerge in the Discovery Environment

Cuffmerge

Comparing wild-type to hy5 transcriptomes

Cuffdiff in the Discovery Environment

Cuffdiff

Cuffdiff Results

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

Density Plot

Scatter Plot

Volcano Plot

Expression Plots

Cloud Computing with iPlant Atmosphere

Launch a Virtual Server (in the Cloud!)

You now have your very own virtual linux server

Expression Plots: Open a terminal and launch R

Expression Plots: Demonstration