TempO-Seq Workflow
Figure 1. TempO-Seq biochemistry and workflow.
BioSpyder Technologies has developed a novel product for targeted sequencing called TempO-Seq™, designed to monitor hundreds to thousands of genes at once in high throughput from as little as 10 pg of total RNA (the amount from a single cell) without pre-amplification, to maximize utilization of precious or limited samples.
Based on BioSpyder Technologies’ proprietary Templated Oligo Detection Assay, TempO-Seq can quantitate targeted transcripts in an easy to follow workflow that does not require dedicated equipment. It can be run in a standard PCR instrument or microplate incubator manually or using standard pipetting platforms. The assay is highly amenable to automation, enabling implementation on 96-, 384-, and 1536-well formats. Sample barcoding, together with sequencing of short templates to measure each gene, allows pooling up to 6,144 samples in one sequencing run. Assay content is flexible and customizable, from focused panels monitoring specific genes or cellular pathways up to the whole transcriptome, delivering unprecedented accuracy and sensitivity for low level inputs. Investigators can select focused content from an archive of detector oligos measuring the whole transcriptome, and add additional custom content such as the measurement of specific isotypes, fusions, or mutations. Together with robust probe design and simplified data analysis that eliminates the need for bioinformatics, TempO-Seq assays deliver an easy to use solution for customers doing expression profiling for any species.
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.
Figure 2. Typical TempO-Seq performance.
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.