ORA

Over-representation analysis (ORA) examines the genes that meet a selection criterion and determines if there are gene sets which are statistically over-represented in that list. This method differs from other methods provided by ErmineJ in that you must set a gene score threshold for gene selection, or define a “hit list” of genes.

Because ORA requires that you set a distinction between “good” and “bad” genes, ORA is most appropriate when you are very confident about the threshold. This is because changing the threshold can change the results, sometimes dramatically. If you are examining genes which naturally fall into two categories (“on chromosome 2” and “not on chromosome 2”), then ORA is the logical choice. Otherwise, in our opinion the other methods are more appropriate.

Technical comment: The probabilities produced by ErmineJ ORA are computed using the hypergeometric distribution, but falls back to using the binomial approximation as needed.

Method overview taken from: http://erminej.msl.ubc.ca/help/tutorials/running-an-analysis-ora

ora(
  scores = NULL,
  hitlist = NULL,
  scoreColumn = 1,
  bigIsBetter = FALSE,
  logTrans = FALSE,
  annotation = NULL,
  aspects = c("Molecular Function", "Cellular Component", "Biological Process"),
  threshold = 0.001,
  geneReplicates = c("mean", "best"),
  pAdjust = c("FDR", "Bonferroni"),
  geneSetDescription = "Latest_GO",
  customGeneSets = NULL,
  minClassSize = 20,
  maxClassSize = 200,
  output = NULL,
  return = TRUE
)

Arguments

scores

A data.frame. Rownames have to be gene identifiers (eg. probes, must be unique), followed by any number of columns. The column used for scoring is chosen by scoreColumn. See http://erminej.msl.ubc.ca/help/input-files/gene-scores/ for information abot how to specify scores. (for test = ORA, GSR and ROC)

hitlist

A vector of gene identifiers. ORA method accepts hitlists instead of scores. If a hitlist is provided, logTrans, thresholds and bigIsBetter options are ignored.

scoreColumn

Integer or character. Which column of the scores data.frame to use as scores. Defaults to first column of scores. See http://erminej.msl.ubc.ca/help/input-files/gene-scores/ for details. (for test = ORA, GSR and ROC)

bigIsBetter

Logical. If TRUE large scores are considered to be higher. FALSE by default (as in p values).

logTrans

Logical. Should the data be -log10 transformed. Recommended for p values. FALSE by default

annotation

Annotation. A file path, a data.frame or a platform short name (eg. GPL127). If given a platform short name it will be downloaded from annotation repository of Pavlidis Lab (https://gemma.msl.ubc.ca/annots/). To get a list of available annotations, use listGemmaAnnotations. Note that if there is a file or folder with the same name as the platform name in the directory, that file will be read instead of getting a copy from Pavlidis Lab. If this file isn't a valid annotation file, the function will fail. If providing a custom annotation file, see makeAnnotation to do it from R or erminej.msl.ubc.ca/help/input-files/gene-annotations/ to do it manually.

If you are providing a custom gene set, you can leave annotation as NULL

aspects

Character vector. Which Go aspects to include in the analysis. Can be in long form (eg. 'Molecular Function') or short form (eg. c('M','C','B'))

threshold

Double. Score threshold (test = ORA only)

geneReplicates

What to do when genes have multiple scores in input file (due to multiple probes per gene)

pAdjust

Which multiple test correction method to use. Can be "FDR" or 'Westfall-Young' (slower).

geneSetDescription

"Latest_GO", a file path that leads to a GO XML or OBO file or a URL that leads to a go ontology file that ends with rdf-xml.gz.

If you left annotation as NULL and provided customGeneSets, this argument is not required and will default to NULL. Otherwise, by default it'll be set to "Latest_GO" which downloads the latest available GO XML file. This option won't work without an internet connection. To get a frozen file that you can use later, see goToday, goAtDate and getGoDates. See http://erminej.msl.ubc.ca/help/input-files/gene-set-descriptions/ for details.

customGeneSets

Path to a directory that contains custom gene set files, paths to custom gene set files themselves or a named list of character strings. Use this option to create your own gene sets. If you provide directory you can specify probes or gene symbols to include in your gene sets. See http://erminej.msl.ubc.ca/help/input-files/gene-sets/ for information about format for this file. If you are providing a list, only gene symbols are accepted.

minClassSize

minimum class size

maxClassSize

maximum class size

output

Output file name.

return

If results should be returned. Set to FALSE if you only want a file

Value

A list containing a "results" component and a "details" component. "results" is a data.frame containing the main output. The columns of this table are

  • Name: the name of the gene set

  • ID: the id of the gene set

  • NumProbes: the number of elements (e.g. probes) in the gene set.

  • NumGenes: the number of genes in the gene set.

  • RawScore: the raw statistic for the gene set. For explanations see this page

  • Pval: the p value for the gene set.

  • CorrectedPvalue: the corrected p pvalue. See this page for more information.

  • MFPvalue: pvalue after multifunctionality correction. Might be missing if correction was not performed.

  • CorrectedMFPvalue: Like CorrectedPvalue, but for the multifunctionality “corrected” pvalue.

  • Multifunctionality: How biased the genes in the set are towards multifunctional genes.

  • Same as: a list of gene sets which have the exact same members as this one. Such gene sets are not listed anywhere else.

  • GeneMembers: If you selected the “Include genes” option when saving, this will contain a list of the genes that are in the gene set, separated by “|”.

"details" section contain settings that were used to run the analysis.