DeepSAGE is a global, digital transcript-profiling technology, which particularly facilitates the identification of rare transcripts [ 33] by producing unique 21 bp cDNA tags (mono-tags) from virtually all mRNA molecules in a sample before high-throughput sequencing.
One of these, digital transcript abundance measurements method is a revolutionary approach for expression analysis competing to replace microarrays for analyzing transcriptome [ 9].
Our digital transcript abundance measurements results showed that 198 genes encoding transcription factors were differentially expressed, including ERF/APETALA2, MYB, and WRKY transcription factors, and MADS-box proteins (Additional file 4).
This enabled direct comparison of digital transcript expression and array hybridization data metrics (Figs. 4 9).
mRNA abundance is determined by sequencing either 3' end tags or random cDNA fragments (digital transcript expression, DTE), followed by read alignment to reference databases and calculation of aligned read frequencies.
Gratifyingly, digital transcript expression values, expressed as aligned read frequencies, were amenable to very similar data transformation, quality control, pattern discovery, row-by-row modeling and annotation analyses as array hybridization datasets.
Abbreviations: DTS, digital transcript subtraction; RDA, representational difference analysis.
To validate the expression patterns revealed by digital transcript abundance measurements results, seven genes identified through digital transcript abundance measurements were analyzed using quantitative real-time PCR.
Quantitative real-time PCR was used to confirm the digital transcript abundance measurements results.
Calyx digital transcript abundance measurements libraries (Table 2) were deep sequenced altogether.
