What microarrays can AltAnalyze work with?
Answer: AltAnalyze can work with nearly any gene expression microarray (e.g., Affymetrix, Illumina, Agilent) as well as RNASeq data. For all array and RNASeq platforms analyzed, AltAnalyze will perform a series of statistical analyses to assess differential gene expression and annotate these results with gene, pathway, Ontology and miRNA binding site predictions (see below). In addition, pathway and ontology over-representation can be automatically performed for differentially expressed genes (user-defined). When analyzing expression data from splicing sensitive platforms (RNASeq, Exon 1.0, Gene 1.0 or Junction arrays), several sophisticated alternative exon analyses are also available (see below). These analyses are also possible for unsupported array platforms (e.g., Affymetrix U133 array), by providing a mapped expression file for each sample (see here).
Supported Gene Expression Analyses
Several useful methods are available for analysis of both conventional and splicing-sensitive platforms in AltAnalyze. These include:
- Expression normalization of CEL files using the RMA algorithm through APT (Affymetrix Only).
- Automated array type identification and download of associated library and annotation files (Affymetrix Only).
- Annotation of probeset-level results with gene annotations (symbol, description), Ensembl IDs, Gene Ontology, WikiPathways, genomic coordinates, microRNA binding site predictions and AltAnalyze custom cellular location (e.g., membrane/extracellular/nuclear) and function (e.g., GPCR subtype, transcript factor) annotations.
- Differential gene expresssion (fold, t-test p-value, f-test p-value, BH False Discovery Rate (FDR) p-values).
- Over-representation analysis using the GO-Elite analysis module.
Supported Alternative Exon Analyses
When analyzing several array and RNASeq platforms, alternative exon detection, annotation and over-representation methods are available. These include:
- Filtering of exon or junction features using either expression or detection p-values (DABG from APT AltAnalyze output). For RNASeq, this includes specific cut-offs for exons and junctions, based on number of read-counts and exon/junction/gene-level RPKMs.
- Alternative exon differential expression using several optional scoring methods.
- Splicing-event, alternative promoter and alternative polyadenylation predictions (available species only) and associated p-value calculation (t-test, f-test, permutation and FDR p-values).
- Comparison of exon-level versus reciprocal-junction results for independent confirmation (RNASeq and JunctionArray).
- Associated alternative isoform prediction (protein-level) and overall sequence differences (e.g., truncation, non-sense mediated decay, alternative N-terminus, alternative coding).
- Protein domain (InterPro) and protein feature (UniProt) composition differences (whole protein versus alternative exon overlapping).
- microRNA binding sites predictions overlapping with alternative-exons.
- Over-representation of alternative exons for: A) pathways, B) alternative domains and C) alternative microRNA binding sites.
- Protein/exon/transcript/domain/pathway visualization in DomainGraph.
For more information, see the full documentation.