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Tolerance Data 2012 Torent

here we consider a set of candidate genes involved in various aspects of the inflammatory response in the liver (the prototypical example of an inflammatory tissue) from the drosophila melanogaster rna-seq data, and quantitatively evaluate the performance of these platforms in the presence of substantial biological variability. for each gene, we define the expression level by calculating the number of reads mapping to that gene from each library, and then normalize these counts to account for library-size variation. we then compare the results of the expression analysis from the two platforms by calculating their spearman correlation coefficient (r). we observe the following:

however, in some cases, the correlation is artificially low due to technical variability in the data. for example, one sample (untreated 9581) consistently has a lower correlation than the others (0.76, 0.7, 0.84, 0.85), indicating that the rna-seq data from this sample is of lower quality than the other samples. this sample also has a significantly lower level of correlation (r = 0.95) when comparing the read counts of the two platforms using the edger transcript quantification pipeline, which we use for all further analyses. this result indicates that the differences in read counts between platforms are most likely driven by biological differences, not by technical ones. the same sample also exhibits significant differences in expression level between the two platforms when comparing libraries from different samples (additional file 10 : table s11).

we determined the expression levels of the genes in clade c and d in two cases: one, in which all samples were analyzed using the same rna extraction, library prep, and sequencing approach (a single experiment), and the second, in which the raw sequencing data were combined across three experiments (combined data). for each of these cases, we also included data from a control experiment that used mixed symbiodinium cultures (i.e., clade a and b), as well as data from the original est study on a. hyacinthus [ 37 ]. we note that, in the case of the combined data, the total number of reads from the different samples was highly correlated across the three experiments (r² = 0.94, p-value = 0.004), but were not perfectly correlated within each experiment. this is consistent with a previous study, which also found that the total number of reads from different samples was highly correlated across technical replicates (correlation coefficient r² = 0.93, p-value < 0.01) [ 25 ].

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