To avoid Type I errors, we normalized the data using a housekeeper gene (see Methods), and we present only those correlations that are significant in both normalized and unnormalized sets of data.
Briefly, both normalized and unnormalized cDNA libraries prepared from purified total RNA derived from the pouch tissues of a single pregnant and non-pregnant seahorse, together with a pool of normalized reference tissues from the pregnant individual (brain, gills, liver, heart, kidney and testes), were individually MID-tagged with a unique sequence identifier.
We performed this analysis for both normalized and un-normalized MI and for differing criteria for Z-score and degree.
Several quality control plots of both the normalized and unnormalized data were generated in the lumi package.
In any case, it is easy to pass between the normalized and unnormalized notions, so we will not worry too much about this point.
The predictor and response variables were associated using imputation techniques based on normalized and unnormalized Euclidean distance, Mahalanobis distance, Independent Component Analysis (ICA), Canonical Correlation Analysis (aka Most Similar Neighbor, or MSN), Canonical Correspondence Analysis (aka Gradient Nearest Neighbor, or GNN), and Random Forest (RF).
Here we present the construction of large normalized EST libraries (Table 1) and assembly and annotation of both normalized and non-normalized libraries.
Similar expression profiles were also observed for each individual library (both normalized and non-normalized), not just the unigene set (data not shown).
For instance, a similar fraction of the genome was assembled from both normalized and non-normalized Illumina libraries with ∼400X coverage, although the normalized library generated 152 fewer contigs (Figure 4a).
2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons.
A total of 132,251 of the DE genes were identical in both, normalized and no-normalized gene sets; 412 DE genes were only identified exclusively in the normalized list and only 6,126 in the no-normalized set of DE genes.
