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cDNA and Small RNA Sequencing

Beckman Coulter Genomics offers several DNA sequencing services that can be initiated from RNA samples. Solutions are available for experimental goals requiring de novo sequencing or re-sequencing of full length transcripts or sensitive tag counting of transcripts or small RNA populations.

Full Length Transcript Applications

De novo Transcript Sequencing

Beckman Coulter Genomics has capably supplied high quality, full length cDNA libraries from total RNA or mRNA samples for de novo transcript sequencing from a wide variety of organisms. Standard, normalized and subtracted cDNA libraries are available and cloned libraries are provided to every customer for every project. Beckman Coulter Genomics has extensive experience decoding transcriptome sequences with Sanger sequencing and more recently has been very successful meeting customer de novo transcript sequencing needs using the 454 Life Sciences* GS FLX Titanium sequencing technology (≥ 350 bp read lengths for cDNA templates). The high-throughput nature of 454 GS FLX Titanium sequencing often eliminates the requirement for cDNA library normalization. Beckman Coulter Genomics has used the 454 GS FLX Titanium to deeply sequence un-normalized cDNA templates from several plant and animal species resulting in de novo assemblies of transcriptomes suitable for use as reference sequences in subsequent experiments.

mRNA-SEQ Transcript Re-sequencing

When a reference transcriptome is available for mapping the mRNA-SEQ protocol on the Illumina* GAIIx sequencing platform is a powerful research tool for transcript identification, quantification and splice variant analysis. Deep sequencing of mRNA-SEQ samples result in sequencing coverage across the length of any transcripts derived from a polyA mRNA species. Mapping results reveal the identity of transcripts present in a sample and the sensitivity of the assay for rare transcripts detection can be increased simply by increasing sequencing depth. In addition to transcript identity the relative expression of exons across a single transcript can elucidate the presence of splice variants of that transcript. Furthermore, the relative expression levels of two transcripts in a single sample or of a single transcript in two disparate samples can be ascertained from relative sequencing depths.

Transcript Tag and Small RNA Counting Applications

Benefits of digital counting powered by Next Generation technologies:

• Dynamic range sensitivity is scalable with increasing sequencing coverage
• No a priori knowledge of target sequences required for experimental design
• Absolute measurements of tag abundance
• Highly reproducible data eliminates the need for technical replicates
• No bias due to hybridization efficiencies

Digital Gene Expression (DGE)

Identifying gene expression levels distinct between a diseased and normal state can be particularly critical to the discovery of pathway alterations that result in a disease phenotype. In DGE, a SAGE*-like technique, tags generated from the 3' ends of cDNAs are identified by sequencing and subsequently quantified. The result is mRNA expression profiles unique to the starting samples. Leveraging the unprecedented throughput of Next Generation sequencing in DGE tag counting enables extraordinary assay sensitivity

Small RNA Sequencing

Beckman Coulter Genomics offers a small RNA sequencing solution using the AB Small RNA Expression Kit (SREK) and the AB SOLiD* sequencing platform to determine small RNA populations, including micro-, short interfering, and piwi interacting RNAs, from a total RNA starting sample. The SREK protocol uniquely conserves DNA strandedness that is identified and reported by the AB SOLiD RNA analysis pipeline.

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