TempO-Seq is unique in its capacity to avoid RNA purification or reverse transcription, by targeting RNAs with detector oligos and removing excess probes and enzymatic inhibitors before the first enzymatic step. Correctly hybridized detector oligos are ligated, then amplified through primer landing sites that are shared among all probes (Figure 1).
This approach permits high target multiplexing because although the central part of the ligated oligos contains diverse sequences, only two PCR primers are needed for any sample, eliminating the primer cross-hybridization and competition inherent in multiplex PCR. Dual sequence tags are incorporated during PCR, to identify up to 6,144 samples in one sequencing library.
Key advantages to TempO-Seq include the capacity to definitively assign correctly ligated products to their RNA targets because the product is sequenced rather than read out on an array. Mis-ligated products can also be detected, unlike on arrays. As a result, and due to BioSpyder optimization efforts, the background reads for no-sample controls are nearly zero. Another advantage to TempO-Seq is that the assay only reports the intended targets. There is no need to eliminate globin or ribosomal RNAs. In addition, the requirement for ligation of two hybridized detector oligos means that the assay demonstrates excellent specificity, with 95% or more single base differential detection. Consequently, the assay selectively measures and discriminates between all members of highly homologous gene families, such as the CYP450. The assay does not require dedicated machinery, and is amenable to automation for high throughput applications.
The assay demonstrates excellent reproducibility (Figure 2), with log2 R2 values routinely exceeding 0.99. The data shown are raw reads, no normalization, for triplicates of total RNA preps from two cell lines (left and center panels). Comparing these cell lines shows dramatic differences in expression, as expected (right panel). Finally, because the sequencing reports the number of ligated probes, the data analysis is simple. Rather than aligning the reads to the genome, TempO-Seq reads are compared to a look up table of ligated detector oligos input to the assay, a task that can be completed on a standard PC within minutes. BioSpyder has developed a data analysis pipeline for users to convert FASTQ files to data tables, with assay quality metrics reported as well, eliminating the need for investigators to have bioinformatics support to perform analysis and generate tables of gene identity versus abundance, or to normalize data between replicates and treatments.
This technology is protected by US Pat. 9856521 and GB 2542929.