Serial analysis of gene expression (SAGE) was a development of EST methodology to increase the throughput of the tags generated and allow some quantitation of transcript abundance. cDNA is generated from the RNA but is then digested into 11 bp "tag" fragments using restriction enzymes that cut DNA at a specific sequence, and 11 base pairs along from that sequence. These cDNA tags are then joined head-to-tail into long strands (>500 bp) and sequenced using low-throughput, but long read-length methods such as Sanger sequencing. The sequences are then divided back into their original 11 bp tags using computer software in a process called deconvolution. If a high-quality reference genome is available, these tags may be matched to their corresponding gene in the genome. If a reference genome is unavailable, the tags can be directly used as diagnostic markers if found to be differentially expressed in a disease state.
The cap analysis gene expression (CAGE) method is a variant of SAGE that sequences tags from the 5’ end of an mRNA transcript only. Therefore, the transcriptional start site of genes can be identified when the tags are aligned to a reference genome. Identifying gene start sites is of use for promoter analysis and for the cloning of full-length cDNAs.
SAGE and CAGE methods produce information on more genes than was possible when sequencing single ESTs, but sample preparation and data analysis are typically more labour-intensive.