{ "metadata": { "name": "Ruphi_OA_RNAseq" }, "nbformat": 3, "nbformat_minor": 0, "worksheets": [ { "cells": [ { "cell_type": "markdown", "metadata": {}, "source": "#Evaluating RNA-seq analysis of Manila Clam larvae experiment\n\nManscript review indicated qPCR was needed to corroborate. Replicating RNA-Seq work to confirm expression differences so target genes can be identified\n\n\n_Updated July 22, 2013 9:50PDT_" }, { "cell_type": "markdown", "metadata": {}, "source": "*from manuscript (methods)*\n\n###RNA-Seq analysis was carried out to determine differential gene expression patterns between the two libraries (ambient and elevated pCO2). RNA-Seq was performed using the following parameters; unspecific match limit = 10, maximum number of mismatches = 2, minimum number of reads = 10. Expression values were measured in RPKM (reads per kilobase of exon model per million mapped reads; see Mortazavi et al., 2008). Differentially expressed genes were identified as having > 1.5 fold difference between libraries and a p-value < 0.10 (Baggerly et al. 2003). Hypergeometric tests on annotations were performed to identify enriched biological processes. This test procedure was performed using CLC Genomics Workbench v4.0 and is similar to the unconditional GOstats test of Falcon and Gentleman (2007). Significantly enriched (p <0.10) GO terms and associated p-values were visualized using REViGO (Reduce + Visualize Gene Ontology) (Supek et al. 2011). ###\n\nand results\n\n###RNA-Seq analysis using Ruphibase as the scaffold identified 3954 differentially expressed contigs. Of those, 162 contigs were expressed at a lower level in larvae exposed to elevated pCO2 conditions, and 3792 were expressed at an elevated level (Figure 3). Among differentially expressed contigs, 204 were expressed over 10-fold higher in elevated pCO2 conditions including the calcification gene Perlucin 6, which was expressed 133 fold higher under elevated pCO2 conditions. Only 8 contigs were expressed 10-fold lower under elevated pCO2 conditions. A complete list of all differentially expressed genes is provided in Supplemental Table 1.###\n" }, { "cell_type": "markdown", "metadata": {}, "source": "In CLC \n\"Screenshot%207/10/13%208:58%20AM\"" }, { "cell_type": "markdown", "metadata": {}, "source": "---\nIssue is numbers do not jive \n\"Screenshot%207/10/13%209:10%20AM\"" }, { "cell_type": "markdown", "metadata": {}, "source": "Seems to be a rounding issue \n\nBelow is screenshot of Supplemental file (that needs work).\n\n\"Screenshot%207/10/13%209:20%20AM\"\n" }, { "cell_type": "code", "collapsed": false, "input": "#Solution is to change text to " }, { "cell_type": "markdown", "metadata": {}, "source": "```\nSELECT\n clc.\"Feature ID\" as CLCID,\n ex.\"Feature ID\" as SuppID,\n ex.\"Target description\",\n ex.\"GO enrichment\",\n ex.\"GO biological process\",\n clc.\"Baggerley's test: 1000 vs 400 original values - P-value\" as CLCp,\n ex.\"P-value\" as SuppP, \n clc.\"Experiment - Fold Change (original values)\" as CLCfold, \n ex.\"Fold Change (original values)\" as SuppFold, \n clc.\"400 - Means\" as CLC400,\n ex.RPKM1 as SuppRPKM1, \n clc.\"1000 - Means\" as CLC1000,\n ex.RPKM as SuppRPKM \n```\n \n\n``` \nFROM [sr320@washington.edu].[table_CLC_Ruphi_400_1000.csv]clc\nleft join [sr320@washington.edu].[table_Ruphi_RNAseq_supp.csv]ex\n on clc.\u200b[Feature ID] = ex.[Feature ID]\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\u200b\n```\n\n\n\n \n--- \nenriched genes \n \n " }, { "cell_type": "markdown", "metadata": {}, "source": "---\n##Comparing qPCR with RNA-Seq\n\nRNA-Seq was carried out using pooled libraries at day 7. Two chambers from each treatment were harvested. August 5th, 2011 was day 7. \nBased on Dave's Notebook it is deduced that for 400ppm, Jars were 4C1 and 4C3. For 1000ppm, -- 1C3 and 1C4.\n\n_Confirmed in Dave's Notebook Dated September 7, 2011._\n\n---\n**CORRECTED DATA** (see below) \n\n\n \n\n \n \n\n\n\"Sam_20121108_150936_2Points.xlsx\"\n\n\n---\n\n\n\"Sam_20121108_150936_2Points.xlsx\"\n\n\n---\n\n\n\"Sam_20121108_161738_2Points.xlsx\"\n\n\n---\n\n\n\"Sam_20121108_161738_2Points.xlsx\"\n\n\n---\n\n\"Sam_20121108_172259_2Points.xlsx\"\n\n\n\n\n\n\n\n\n---\n**EXCEL ERROR - ALL DATA BELOW WRONG** \n\nSam did qPCR on select genes, data @\n\nhttp://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_172259.xlsx\n\nhttp://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_161738.xlsx\n\nhttp://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_150936.xlsx\n\n\n---\n\n\"Screenshot%207/17/13%207:44%20AM\" \n\n---\n\"Screenshot%207/17/13%207:42%20AM\"\n\n---\n\"Sam_20121108_150936.xlsx\"\n" }, { "cell_type": "code", "collapsed": false, "input": "", "language": "python", "metadata": {}, "outputs": [] } ], "metadata": {} } ] }