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tutorial [2013/03/30 11:03] sotacam |
tutorial [2014/01/08 11:55] (current) sotacam |
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| ====== NOISeq Tutorial ====== | ====== NOISeq Tutorial ====== | ||
| - | The NOISeq tutorial can be downloaded from Bioconductor website. Click [[http://www.bioconductor.org/packages/2.12/bioc/html/NOISeq.html|here]]. | + | The NOISeq tutorial can be downloaded from Bioconductor website. Click [[http://www.bioconductor.org/packages/release/bioc/html/NOISeq.html|here]]. |
| - | In NOISeq package, you can find not only the method to compute **differential expression** between two experimental conditions from RNA-seq data, but also other functions in order to learn more about saturation, contamination or other biases in your data as well as to explore the differential expression results. | + | In NOISeq package, you can find not only the methods NOISeq and NOISeqBIO to compute **differential expression** between two experimental conditions from RNA-seq data, but also other functions in order to learn more about saturation, contamination or other biases in your data as well as to explore the differential expression results. |
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| - | ====== NOISeqBIO Tutorial ====== | ||
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| - | Please, follow these instructions to run NOISeqBIO: | ||
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| - | **1)** Install NOISeq Bioconductor package (see [[downloads|Downloads]]) and load it: ''library(NOISeq)''. | ||
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| - | **2)** Download the zip file containing the R scripts for NOISeqBIO (from [[downloads|Downloads]]) and unzip it. | ||
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| - | **3)** To load the NOISeqBIO functions in R from your working directory: | ||
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| - | # Remember you have to change this path if you are not in the same directory: | ||
| - | > mypath = "NOISeqBIO_v00" | ||
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| - | > source(file.path(mypath, "auxiliar.R")) | ||
| - | > source(file.path(mypath, "MDbio.R")) | ||
| - | > source(file.path(mypath, "allMDbio.R")) | ||
| - | > source(file.path(mypath, "noiseqbio.R")) | ||
| - | > source(file.path(mypath, "classes.R")) | ||
| - | > source(file.path(mypath, "normalization.R")) | ||
| - | > source(file.path(mypath, "fewreplicates.R")) | ||
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| - | **4)** Read your data as in NOISeq (see the following example with Marioni's data, where liver and kidney tissues are compared) to convert them to a NOISeq object: | ||
| - | > data(Marioni) | ||
| - | > mydata = readData(data = mycounts, factors = myfactors) | ||
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| - | **5)** To apply NOISeqBIO with TMM normalization (you can choose the same normalization procedures as in NOISeq): | ||
| - | > mynoiseq.test = noiseqbio(mydata, norm = "tmm", factor = "Tissue", r = 10) | ||
| - | The parameter //r// is the number of permutations of sample labels to generate null distribution. | ||
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| - | **6)** Finally, select the differentially expressed genes: | ||
| - | # Probability cutoff. It is equivalent to a FRD (adjusted p-value) of 0.05 | ||
| - | > myq = 0.95 | ||
| - | > mydeg = mynoiseq.test@results[[1]][which(mynoiseq.test@results[[1]][,"prob"] > myq),] | ||
| - | > nrow(mydeg) | ||
| - | [1] 2837 | ||
| - | > head(mydeg) | ||
| - | Kidney Liver theta prob | ||
| - | ENSG00000187642 12.8606633 4.332902 0.8107475 0.9601231 | ||
| - | ENSG00000188290 17.7544458 4.961376 1.3160369 0.9999999 | ||
| - | ENSG00000187608 19.2702201 34.329320 -0.7153955 0.9543329 | ||
| - | ENSG00000188157 691.1874857 141.575959 5.9237953 1.0000000 | ||
| - | ENSG00000186891 0.8230924 2.736435 -0.7348520 0.9568449 | ||
| - | ENSG00000078808 372.5016774 483.708484 -1.0066939 0.9787270 | ||
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| - | Unfortunately, the DE plots in NOISeq package cannot be used yet on these DE results. Please, find below how to draw a plot to illustrate the DE results: | ||
| - | > cond1 = log2(rowMeans(mycounts[,grep("Kidney", colnames(mycounts))]) + 1) | ||
| - | > cond2 = log2(rowMeans(mycounts[,grep("Liver", colnames(mycounts))]) + 1) | ||
| - | > plot(cond1, cond2, cex = 0.7, xlab = "Kidney (Average expression in log-scale)", | ||
| - | ylab = "Liver (Average expression in log-scale)", main = "NOISeqBIO on Marioni's data") | ||
| - | > points(cond1[rownames(mydeg)], cond2[rownames(mydeg)], pch = 20, col = "red", cex = 0.5) | ||
tutorial.1364637781.txt.gz ยท Last modified: 2013/03/30 11:03 by sotacam
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