• Document: Whole Transcriptome Analysis of Illumina RNA- Seq Data. Ryan Peters Field Application Specialist
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Whole Transcriptome Analysis of Illumina RNA- Seq Data Ryan Peters Field Application Specialist Partek® GS™ in your NGS Pipeline Your Start-to-Finish Solution for Analysis of Next Generation Sequencing Data Data RNA-Seq / SmallRNA- Seq ChIP-Seq DNA-Seq MeDIP-Seq coming soon 2 Copyright © Partek Inc All rights reserved Partek Web Tools (coming soon) 3 Copyright © Partek Inc All rights reserved Data Import Sequence Reads Flexible import Many supported formats Align Reads to Reference Genome 4 Copyright © Partek Inc All rights reserved RNA-Seq workflow for Whole Transcriptome Analysis • Import & Quality Control • Assign reads to known RNAs in a transcriptome db • Differential expression of mRNAs • Identification of alternative splicing events • Differential expression of non-coding RNAs • Coding SNP discovery • Biological Interpretation • Detect Unexplained Regions 5 Copyright © Partek Inc All rights reserved Biological Replicates? • 2 Illumina RNA-Seq Datasets • Dataset #1 • Breast Cancer • Biological Replicates - YES • Expression of genes between ER+, ER-, Normal • Dataset #2 • Brain vs. UHR • Biological Replicates - NO • Expression profile of genes between Brain sample and UHR sample • Goal: Understand different types of statistical tests used for each scenario 6 Copyright © Partek Inc All rights reserved Assign reads to known isoforms– modified E/M algorithm • Junction reads • Paired end reads • Multiple aligned reads • Strand-specific reads • Strand-specific reads can distinguish genes transcribed from forward/reverse strand. 7 Copyright © Partek Inc All rights reserved Dataset #1 Breast Cancer Data w/ Replicates Breast Cancer Dataset – Illumina’s iDEA Data Set • Illumina iDEA challenge (Illumina Data in Excellence Award) • 8 Paired-End RNA-Seq samples • 4 ER +, 3 ER -, 1 Normal Control – Breast Cancer Cell Lines • Replicates • Aligned using TopHat, junction alignment available • BAM format 9 Copyright © Partek Inc All rights reserved Summary Report Know where your reads are mapping: 10 Copyright © Partek Inc All rights reserved Principal Components Analysis(PCA) 11 Copyright © Partek Inc All rights reserved Biological Replicates - ANOVA Most powerful ANOVA implementation in Partek GS 1. Balanced, Unbalanced & Incomplete 2. Random & Fixed Effects (mixed model) 3. Nested Hierarchical designs 4. Numeric & Categorical Variables 5. Any number of factors 6. Linear Contrasts 12 Copyright © Partek Inc All rights reserved Statistical Report 13 Copyright © Partek Inc All rights reserved Create Gene List 14 Copyright © Partek Inc All rights reserved Hierarchical Clustering of Significant Genes 15 Copyright © Partek Inc All rights reserved GO Enrichment 16 Copyright © Partek Inc All rights reserved Visualize Diff. Expression and Alternative Splicing 17 Copyright © Partek Inc All rights reserved Allele Specific Expression Use Analysis of Variance to study allele specific expression based on the interaction of allele (A, T, G, C) counts and sample

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