2/3/2015 1 HMGP 7620: Advanced Genome Analysis Quantitative RNA Sequencing (RNA-seq) and Exome Analysis Richard A. Radcliffe, Ph.D. Professor of Pharmacology School of Pharmacy, Department of Pharmaceutical Sciences Room V20-3124 (303) 724-3362 [email protected]Why RNA-seq? Crick (1970) Nature 227:561-563 Phenotype Genetic architecture Developmental stage Environmental influences Tissue type Disease state HMGP 7620: Advanced Genome Analysis
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Quantitative RNA Sequencing (RNA-seq) and Exome … 1 HMGP 7620: Advanced Genome Analysis Quantitative RNA Sequencing (RNA-seq) and Exome Analysis Richard A. Radcliffe, Ph.D. Professor
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2/3/2015
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HMGP 7620: Advanced Genome Analysis
Quantitative RNA Sequencing (RNA-seq) and Exome Analysis
Richard A. Radcliffe, Ph.D.Professor of Pharmacology
School of Pharmacy, Department of Pharmaceutical SciencesRoom V20-3124(303) 724-3362
“Understanding the transcriptome is essential for interpreting the functional elements of the genome and revealing the molecular
constituents of cells and tissues, and also for understanding development and disease.”
• Catalogue all species of transcript, including mRNAs, non-coding RNAs and small RNAs
• Determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications
• Quantify the changing expression levels of each transcript during development and under different conditions.
• Pathway/network/ontology analysis.
Why RNA-seq?
Massively parallel expression analysis
Wang et al. (2009) Nat Rev Genetics 10:57-63HMGP 7620: Advanced Genome Analysis
RNA-seq OverviewAAAAAA AAAAAA
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AAAAAA AAAAAA
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AAAAAA AAAAAAAAAAAA AAAAAA AAAAAA
AAAAAA AAAAAA
Adapted from: Pepke et al. (2009) Nat Methods 6:S22-S32
Analysis(QC, quantitation, transcript
annotation)
Select fraction of interest
Library prep
Sequence and map to reference genome
HMGP 7620: Advanced Genome Analysis
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Library Prep
HMGP 7620: Advanced Genome Analysis Corney (2013) Mater Methods 3:203
Library Prep: Some Considerations
HMGP 7620: Advanced Genome Analysis
• RNA fraction – Many different RNA species– Poly(A)– Size (<200 nt vs. >200 nt)
• Strandedness• Read length• Single- vs. pair-end• Multiplexing
• RNA fraction – Many different RNA species– Poly(A)– Size (<200 nt vs. >200 nt)
• Strandedness– Overlapping transcripts– Annotation of novel transcripts
• Read length• Single- vs. pair-end • Multiplexing
Library Prep: Some Considerations
Slide 7
RR34 The area of the box represents the genome. The area of large green circle is equivalent to the documented extent of transcription, with the darker green area corresponding to that on both strands. CDSs are protein-coding sequences, and UTRs are 5′- and 3′-untranslated sequences in mRNAs. The dots indicate (and in fact overstate) the proportion of the genome occupied by known snoRNAs and miRNAs. Richard Radcliffe, 1/26/2015
No question about which strand(gene) the fragment came from.
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HMGP 7620: Advanced Genome Analysis
• RNA fraction – Many different RNA species– Poly(A)– Size (<200 nt vs. >200 nt)
• Strandedness• Read length• Single- vs. pair-end• Multiplexing
Library Prep: Some Considerations
Read Length
HMGP 7620: Advanced Genome Analysis
• Read length is related to:– Sequencing accuracy: quality declines as a function of the length of a read– Mapping accuracy: the longer the read, the more accurately it maps
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HMGP 7620: Advanced Genome Analysis
• RNA fraction – Many different RNA species– Poly(A)– Size (<200 nt vs. >200 nt)
• Strandedness• Read length• Single- vs. pair-end • Multiplexing
RR1 Examples of intragenic deletion and duplication detected by WES and confirmed by exome aCGH. Each bar in the graphs (a)–(c) and (e)–(g) represents an exon. (a–c) WES data from a family trio in which the (a) proband has inherited a whole-gene duplication of KRT34 from the (b) father, whereas the (c) mother shows normal copy number at that gene. (e–g) WES data from a family trio in which the (e) proband has inherited a partial-gene heterozygous deletion in the SYCP2L gene from the (g) mother, whereas the (f) father shows normal copy number at those exons. Each dot in panels d and h represents an oligonucleotide probe in the gene of interest on the exome array, with a duplication shown by probes deviating to a positive log2 ratio (marked in red) and a deletion shown by probes deviating to a negative log2 ratio (marked in green). Panels d and h show confirmation of the KRT34 duplication and the SYCP2L deletion, respectively, by exome aCGH. aCGH, array comparative genomic hybridization; WES, whole-exome sequencing.Radcliffe, Richard, 2/1/2015
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Variant Calling: CNVs/Indels
HMGP 7620: Advanced Genome Analysis Retterer et al. (2014) Genetics Med doi:10.1038/gim.2014
Child
Father
Mother
RR2
Genetic Analysis: Mendelian Inheritance
HMGP 7620: Advanced Genome Analysis
Assumptions:• Only consider small indels and
SNPs• Causal variants are coding• Causal variants alter protein
sequence• Near complete penetrance
Rabbani et al. (2012) J Hum Genetics 57:621-632
Slide 41
RR2 Examples of intragenic deletion and duplication detected by WES and confirmed by exome aCGH. Each bar in the graphs (a)–(c) and (e)–(g) represents an exon. (a–c) WES data from a family trio in which the (a) proband has inherited a whole-gene duplication of KRT34 from the (b) father, whereas the (c) mother shows normal copy number at that gene. (e–g) WES data from a family trio in which the (e) proband has inherited a partial-gene heterozygous deletion in the SYCP2L gene from the (g) mother, whereas the (f) father shows normal copy number at those exons. Each dot in panels d and h represents an oligonucleotide probe in the gene of interest on the exome array, with a duplication shown by probes deviating to a positive log2 ratio (marked in red) and a deletion shown by probes deviating to a negative log2 ratio (marked in green). Panels d and h show confirmation of the KRT34 duplication and the SYCP2L deletion, respectively, by exome aCGH. aCGH, array comparative genomic hybridization; WES, whole-exome sequencing.Radcliffe, Richard, 2/1/2015
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Genetic Analysis
HMGP 7620: Advanced Genome Analysis Ku et al. (2012) Ann Neurology 71:5-14
A Few ReferencesRNA-seq:• Griffith M, Griffith OL, Mwenifumbo J, Goya R, Morrissy AS, Morin RD, Corbett R, Tang MJ, Hou YC, Pugh TJ, Robertson G,
Chittaranjan S, Ally A, Asano JK, Chan SY, Li HI, McDonald H, Teague K, Zhao Y, Zeng T, Delaney A, Hirst M, Morin GB, Jones SJ, Tai IT, Marra MA (2010) Alternative expression analysis by RNA sequencing. Nat Methods 7:843-847.
• Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621-628.
• Munger SC, Raghupathy N, Choi K, Simons AK, Gatti DM, Hinerfeld DA, Svenson KL, Keller MP, Attie AD, Hibbs MA, Graber JH, Chesler EJ, Churchill GA (2014) RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations. Genetics 198:59-73.
• Oshlack A, Robinson MD, Young MD (2010) From RNA-seq reads to differential expression results. Genome Biol 11:220.
• Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57-63.
Exome sequencing:• Altmann A, Weber P, Bader D, Preuß M, Binder E, Müller-Myhsok B (2012) A beginners guide to SNP calling from high-
• Biesecker LG, Green RC (2014) Diagnostic clinical genome and exome sequencing. The New England Journal of Medicine370:2418-2425.
• Krumm N, Sudmant PH, Ko A, O'Roak BJ, Malig M, Coe BP, Quinlan AR, Nickerson DA, Eichler EE (2012) Copy number variation detection and genotyping from exome sequence data. Genome Res 22:1525-1532.
• Majewski J, Schwartzentruber J, Lalonde E, Montpetit A, Jabado N (2011) What can exome sequencing do for you? Journal of Medical Genetics 48:580-589.
• Singleton AB (2011) Exome sequencing: a transformative technology. The Lancet Neurology 10:942-946.