An Introduction to RNA-Seq Transcriptome Profiling with iPlant (

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:

Samples Experimental data downloaded from the NCBI Short Read Archive (GEO:GSM and GEO:GSM613466) Two replicates each of RNA-seq runs for Wild- type and hy5 mutant seedlings.

RNA-Seq Conceptual Overview Image source:

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 runWT-1WT-2hy5-1hy5-2 Reads10,866,70210,276,26813,410,01112,471,462 Seq. (Mbase)

RNA-Seq HWUSI-EAS455:3:1:1:1096 length=41 CAAGGCCCGGGAACGAATTCACCGCCGTATGGCTGACCGG C HWUSI-EAS455:3:1:2:1592 length=41 GAGGCGTTGACGGGAAAAGGGATATTAGCTCAGCTGAATCT + @SRR HWUSI-EAS455:3:1:2:869 length=41 TGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCA + HWUSI-EAS455:3:1:4:1075 length=41 CAGTAGTTGAGCTCCATGCGAAATAGACTAGTTGGTACCAC HWUSI-EAS455:3:1:5:238 length=41 AAAAGGGTAAAAGCTCGTTTGATTCTTATTTTCAGTACGAA + @SRR HWUSI-EAS455:3:1:5:1871 length=41 GTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTGTAAG HWUSI-EAS455:3:1:5:1981 length=41 GAACAACAAAACCTATCCTTAACGGGATGGTACTCACTTTC + : …Now What?

@SRR HWUSI-EAS455:3:1:1:1096 length=41 CAAGGCCCGGGAACGAATTCACCGCCGTATGGCTGACCGG C HWUSI-EAS455:3:1:2:1592 length=41 GAGGCGTTGACGGGAAAAGGGATATTAGCTCAGCTGAATCT + @SRR HWUSI-EAS455:3:1:2:869 length=41 TGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCA + HWUSI-EAS455:3:1:4:1075 length=41 CAGTAGTTGAGCTCCATGCGAAATAGACTAGTTGGTACCAC HWUSI-EAS455:3:1:5:238 length=41 AAAAGGGTAAAAGCTCGTTTGATTCTTATTTTCAGTACGAA + @SRR HWUSI-EAS455:3:1:5:1871 length=41 GTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTGTAAG HWUSI-EAS455:3:1:5:1981 length=41 GAACAACAAAACCTATCCTTAACGGGATGGTACTCACTTTC + : Bioinformatician

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 Discovery Environment Atmosphere

The iPlant Discovery Environment

Staged Data

Tophat

TopHat in the Discovery Environment

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.

Assembling the Transcripts

Cufflinks in the Discovery Environment

Merging the Transcriptomes

Cufffmerge in the Discovery Environment

Comparing wild-type to hy5 transcriptomes

Cuffdiff in the Discovery Environment

Cuffdiff Results

Differentially expressed genes Example filtered Cuffdiff results generated in the Discovery Environment.

Differentially expressed transcripts Example filtered Cuffdiff results generated in the Discovery Environment.