• Document: RNA spike-in controls & analysis methods for trustworthy genome-scale measurements
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RNA spike-in controls & analysis methods for trustworthy genome-scale measurements Sarah A. Munro, Ph.D. Genome-Scale Measurements Group ABRF Meeting March 29, 2015 Overview • External RNA Controls Consortium (ERCC) RNA spike-in controls • ‘erccdashboard’ analysis tool • ERCC 2.0: Building an updated suite of RNA controls Overview • External RNA Controls Consortium (ERCC) RNA spike-in controls • ‘erccdashboard’ analysis tool • ERCC 2.0: Building an updated suite of RNA controls How can we have trustworthy gene expression results? • We’re simultaneously measuring thousands of RNA molecules in gene expression experiments • But are we getting it right? External RNA Controls Consortium (ERCC) initiated by industry, hosted by NIST • Initiated by Janet Warrington, VP Clinical Genomics at Affymetrix • Open to all interested parties Spike- • Voluntary ins • More than 90 participants – Industry, Academia, Government – All major microarray technology developers – Other gene expression assay developers ERCC control sequences are in NIST Standard Reference Material 2374 • DNA sequence library • 96 unique control sequences in DNA plasmids • Controls intended to mimic mammalian mRNA • In vitro transcription to make RNA controls NIST SRM 2374 and related data files are available directly from NIST @ http://tinyurl.com/erccsrm Making ERCC ratio mixtures with true positive and true negative ratios NIST Plasmid RNA transcripts Mixtures with known DNA Library abundance ratios in vitro Pooling … transcription Using ERCC ratio mixtures Treated (n>3) Control (n>3) Using ERCC ratio mixtures Treated (n>3) Control (n>3) Using ERCC ratio mixtures Treated (n>3) Control (n>3) Using ERCC ratio mixtures Treated (n>3) Control (n>3) Measurement process Multiple steps Many people & labs Expression Measures Takes days to weeks Statistical Analysis Example gene expression data Treated Control Are the RNA molecule ratios statistically different across the samples? Treated Control Evaluate technical performance with ERCC true positive and true negative ratios Treated Control Overview • External RNA Controls Consortium (ERCC) RNA spike-in controls • ‘erccdashboard’ analysis tool • ERCC 2.0: Building an updated suite of RNA controls Use erccdashboard to produce standard performance metrics for any experiment • R package is available from: – Bioconductor – NIST GitHub Site • Open source and open access for use in – Other analysis tools and pipelines – Commercial software Gauge technical performance with 4 erccdashboard figures • Developed as part of SEQC study, with ABRF partners • Technology-independent ratio performance measures • Assessed differences in performance across – Experiments – Laboratories – Measurement processes Munro, S. A. et al. Nature Communications 5:5125 doi: 10.1038/ncomms6125 (2014). Ambion ERCC Ratio Mixtures 23 Controls per Subpool Design abundance spans 220 range within each Subpool Spike-in design for SEQC RNA Sequencing Experiments Samples replicates for sequencing Rat Experiment Interlaboratory Experiment Treated and Control Rat RNA Human Reference RNA Samples Biological Replicates Technical Replicates What is the dynamic range of my experiment? Rat Experiment Interlaboratory Experiment Log2 Normalized ERCC Counts Log2 Normalized ERCC Counts Log2 ERCC Spike Amount (attomol nt µg-1 total RNA) Log2 ERCC Spike Amount (attomol nt µg-1 total RNA) What is the dynamic range of my experiment? Rat Experiment Interlaboratory Experiment Typical Deep Sequencing

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