Gene Expression Profiling – RNA, Whole Transcriptome Sequencing and Digital Gene Expression

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October 2nd 2012


Gene expression profiling compares the expression levels of thousands of genes at once and provides an insight into the characterization of molecular changes.

VERTIS offers the following gene expression profiling services:

Sequencing-based RNA expression profiling
VERTIS has extensive expertise in next generation sequencing the leading tool for genetic and genomic analysis. With the introduction of the new NGS methods it is now possible to perform rapid and highly reproducible analysis of complete transcriptional networks by generating millions of – depending on the platform – 35 to 400 bp long reads.

A typical eukaryotic transcriptome is composed of approximately 20,000 different transcripts with an average length of 1,000 nucleotides (nt), together making a total of about 20 million nucleotides. In a single GS FLX Titanium run approximately 1 million reads with an average length of 350 bp are generated – a total of 350 million nucleotides – therefore, in a single GS FLX run, a human transcriptome can be captured in a more than 17-fold coverage.

VERTIS determines gene expression profiles by sequencing of defined cDNA tags, derived from the 3′-ends of the transcripts, or by whole transcriptome sequencing.

3′ Tag Sequencing
Sequencing of 3′-fragment cDNA which starts 100 – 400 bp upstream of the polyadenylation sites of the transcripts. Because each transcript is represented by only a single tag, 3′-tag libraries are substantially less complex than standard RNA-Seq libraries, allowing useful results with fewer reads per sample, thus making 3’tag sequencing much more cost efficient. Because of the large number of reads generated, the best choice for differential expression analysis using sequencing of short 3′ sequence tags is the use of the Illumina HiSeq sequencer.

Similarly, sequencing of sncRNA with lengths of 20-30 nt is best performed with an Illumina sequencer.

Whole Transcriptome Sequencing
Sequencing of random-primed cDNA which covers the full-length of the transcripts. The method allows gathering of both, genome wide sequence information and comprehensive gene expression data. Best performed, especially in de-novo sequencing projects, with the Roche GS FLX platform – the long reads generated (350 bp in average) strongly facilitate the assembly of the cDNA fragments into long contigs which cover the transcripts in full length.

Advantage of Digital Gene Expression
In comparison with microarrays, digital gene expression increases the dynamic range of analyses, has the ability to detect novel exons and does not have to deal with false positives – thus allowing accurate, comprehensive and highly reproducible quantification of gene expression.

Delivered Data
Expression data are obtained by mapping the bar-coded reads against the reference genome or an existing contig file.

For more information or to discuss gene expression profiling please contact us directly.