In order to identify miRNAs independently of their miRBase annotation, we applied the miRDeep algorithm (Friedländer et al. 2008) to our data set.
> -wrap-foot> Retrieval of samples with similar annotation: We applied the proposed approach and a number of alternatives for retrieval of similar samples from the same dataset and evaluated by how many of the retrieved samples had the same annotation: class label, disease state or body site.
For functional annotation, we applied Ingenuity Pathways Analysis Ingenuity Systemss, ) and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) [ 34].
In a second analysis using the GO annotations, we applied the GO term 'ATPase activity, coupled to transmembrane movement of substances' (GO 042626).
In designing the k-mer frequency approach to be completely independent of any manual annotation, we apply a binary decision framework: given a numerical threshold, sequence is designated as either repeat or non-repeat.
To provide estimates of the accuracy of functional modules on inferring reliable functional annotations, we applied a cross-validation procedure to predict functional annotations (see methods).
To have high quality of gene start annotation, herein we applied the ProTISA pipeline for gene start relocation of the CFT073 genome [ 22].
By manually checking the annotations we noticed inconsistency in the way the annotators applied the annotation guidelines along this dimension, leading to fewer than 100 examples for each of the negative and the positive trends, and performance, as expected, deteriorates.
The annotation strategy applied to the newly sequenced mtDNAs was based on the guidelines provided by Lavrov et al. (2000).
Of these, 64 are due to annotation differences and are found in the PSU-1 genome via the annotation pipeline applied in this study compared to that used in the original annotation of the PSU-1 genome.
However, the over-representation analysis at the functional level that we applied here allows multiple annotations for a single gene and, therefore, inherently overcomes this type of problems.
