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Published byBethany Small Modified over 9 years ago
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Genomics I: The Transcriptome RNA Expression Analysis Determining genomewide RNA expression levels
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Genome-wide expression analysis methods Microarrays –Hybridization based RNA-seq –Direct sequencing of cDNAs
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RNA- seq High throughput sequencing of cDNAs from biological samples Determine abundance of mRNA by representation in sequencing reads May detect variants (alternative splicing, specific alleles, etc.)
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DNA Sequencing Cost per Genome
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Comparison of NextGen Sequencing Platforms
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A decade’s perspective on DNA sequencing technologyA decade’s perspective on DNA sequencing technology Elaine R. Mardis Nature 470, 198–203 (10 February 2011) doi:10.1038/nature09796 Elaine R. Mardis
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Post-sequencing Analysis Align seq reads to each other or to reference genome Score number of reads per transcript (abundance) Compare abundance between experimental groups (statistical analysis of difference)
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Analysis of transcriptome data Inherent variability: need for repetition –Biological and technical replicates Analysis algorithms –Based on statistical models Requires verification by independent methods –Quantitative PCR (Real Time RT-PCR) Means of generating hypotheses that need to be tested
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Real-time PCR Sensitive means of measuring RNA abundance Not genomewide: used to verify microarray results SYBR Green method: Dye binds only to double- stranded nucleic acid and fluoresces when bound Fluorescence is proportional to amount of double-stranded DNA
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Real-time PCR readout The readout of a real- time PCR reaction is a set of curves The curves indicate the PCR cycle at which fluorescence is detected Each cycle is twice the amount of the previous cycle
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Meta-analysis of Microarray Data
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