Shambhala-2: A Protocol for Uniformly Shaped Harmonization of Gene Expression Profiles of Various Formats

Uniformly shaped harmonization of gene expression profiles is central for the simultaneous comparison of multiple gene expression datasets. It is expected to operate with the gene expression data obtained using various experimental methods and equipment, and to return harmonized profiles in a uniform shape. Such uniformly shaped expression profiles from different initial datasets can be further compared directly. However, current harmonization techniques have strong limitations that prevent their broad use for bioinformatic applications. They can either operate with only up to two datasets/platforms or return data in a dynamic format that will be different for every comparison under analysis. This also does not allow for adding new data to the previously harmonized dataset(s), which complicates the analysis and increases calculation costs. We propose here a new method termed Shambhala-2 that can transform multi-platform expression data into a universal format that is identical for all harmonizations made using this technique. Shambhala-2 is based on sample-by-sample cubic conversion of the initial expression dataset into a preselected shape of the reference definitive dataset. Using 8390 samples of 12 healthy human tissue types and 4086 samples of colorectal, kidney, and lung cancer tissues, we verified Shambhala-2's capacity in restoring tissue-specific expression patterns for seven microarray and three RNA sequencing platforms. Shambhala-2 performed well for all tested combinations of RNAseq and microarray profiles, and retained gene-expression ranks, as evidenced by high correlations between different single- or aggregated gene expression metrics in pre- and post-Shambhalized samples, including preserving cancer-specific gene expression and pathway activation features. © 2022 Wiley Periodicals LLC. Basic Protocol: Shambhala-2 harmonizer Alternate Protocol 1: Linear Shambhala/Shambhala-1 Alternate Protocol 2: Alternative (flexible-format and uniformly shaped) normalization methods Support Protocol 1: Watermelon multisection (WM) Support Protocol 2: Calculation of cancer-to-normal log-fold-change (LFC) and pathway activation level (PAL).