Limitations of RNA-Seq

Gene expression studies can often benefit from testing large numbers of samples, obtaining repeatable as well as accurate results and from low sequencing costs/sample, and low analysis costs. Analysis platforms have evolved from outdated hybridization array based technologies to RNA-Seq, and quantitative real time reverse transcription PCR (qRT-PCR) has evolved into highly multiplexed platforms. Though RNA-Seq has become a gold standard and can be used as a quantitative assay to determine relative transcript abundance, it is costly, onerous, and employs a time-intensive process for assay design, running the assay and data analysis.

Generating libraries for mRNA sequencing is a difficult and often error prone process involving many steps with loss of sample at every step. The RNA must be extracted and reverse transcribed, then processed further to generate the sequencing library. The presence of high abundance RNAs (rRNA, etc) requires additional steps to reduce background RNA and/or enrich for mRNAs. Although these methods can help data quality, they add to the labor, cost and time required and deplete the amount of original sample, which is especially problematic when working with needle biopsies, rare transcripts or single cells. To address these issues, many resort to pre-amplification of the RNA as well as to deeper sequencing to increase the number of reads. This presents challenges to data analysis, reduces the number of samples that can be batched together in a single library, and increases both cost per sample and time.

RNA-Seq is best used to identify new biomarkers, gene variants, or mutations, but is especially inefficient if gene markers have already been identified and consequently all the information of interest comes from this focused subset of genes. While qRT-PCR based methods are perfectly acceptable when measuring low numbers of targets, they are impractical when large numbers of targets need to be analyzed with high throughput sample processing, and like RNA-Seq, require RNA to be extracted and reverse transcribed. Multiplexing the measurement of more than one gene at a time within the same PCR reaction requires extensive optimization, and is limited to at most 4 genes at a time in any given reaction. Microfluidic and microplate platforms are available that permit a sample to be split across multiple PCR reactions for many different genes, but when configured, for instance, to measure 96 samples across 96 genes, the cost per sample is very high and the amount of sample is limiting.

Targeted sequencing methods have been developed that range from capturing a subset of targeted genes on an array and then releasing and sequencing these, to use of targeted PCR primers or reverse transcriptase primers to selectively amplify and process subsets of targeted genes. However, primer amplification-based methods of targeted sequencing have proven difficult to develop for a set of primers for each target, and as a result content is typically limited to 500 to 1000 genes. These approaches still require extraction and reverse transcription.

These serious limitations of qPCR, RNA-Seq, and targeted sequencing methods have driven the need for a higher throughput, higher content, simpler, and more sensitive targeted sequencing approach that is not limited in the number of genes that can be measured, or by the complexity of developing assays with different content, and which reduces the complexity of the transcriptome to short read sequences for each targeted gene.

To address these challenges and enable high-throughput, low-cost screening, Biospyder has developed TempO-Seq™.