We established a colonic adenoma-normal mucosa suppressive subtraction hybridization (SSH) library in 1999. In this study, we wanted to explore the expression profile of all candidate genes in this library. We developed an EST pipeline which contained two in-house software packages, nucleic acid analytical software and GetUni. The nucleic acid analytical software, an integrator of the universal bioinformatics tools including phred, phd2fasta, cross_match, repeatmasker and blast2.0, can blast sequences of differential clones with the downloaded non-redundant nucleotide (NR) database. GetUni can cluster these NR sequences into Unigene via matching with the downloaded Homo Sapiens UniGene database. Sixty-two candidate genes in A-N library were obtained via the high throughput automatic gene expression bioinformatics pipeline. Gene Ontology online analysis revealed that ribosome genes and immunity-regulating genes were the two most common categories in the KEGG or Biocarta Pathway. We also detected the expression of 2 genes with highest hits, Reg4 and FAM46A, by semi-quantitative RT-PCR. Both genes were up-regulated in 10 or 9 out of 10 adenomas in comparison with the paired normal mucosa, respectively. The candidate genes in A-N library would be of great significance in disclosing the molecular mechanism underlying in colonic adenoma initiation and progression.