title: SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea.
abstract: BACKGROUND
  Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process.
  
  
  RESULTS
  Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots.
  
  
  CONCLUSIONS
  We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from http://microarray.up.ac.za/SSHscreen.
authors.affiliation: ACGT Computational Biology and Bioinformatics Unit, Department of Biochemistry, University of Pretoria, 0002, South Africa.
authors.forename: Nanette
authors.initials: N
authors.lastname: Coetzer
authors.affiliation: Germplasm Development Division, Agricultural Research Council-Vegetable and Ornamental Plant Institute, Private Bag X293, Pretoria, 0001, South Africa.|Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, 0002, South Africa.
authors.forename: Inge
authors.initials: I
authors.lastname: Gazendam
authors.affiliation: Germplasm Development Division, Agricultural Research Council-Vegetable and Ornamental Plant Institute, Private Bag X293, Pretoria, 0001, South Africa.
authors.forename: Dean
authors.initials: D
authors.lastname: Oelofse
authors.affiliation: Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, 0002, South Africa.
authors.forename: Dave K
authors.initials: DK
authors.lastname: Berger