INTRODUCTION
BioClavis is offering a proprietary high-throughput TempO-LINC (Ligation INdexed Cells) single-cell genomics platform. This product relies on the foundational TempO-Seq technology approach that BioClavis developed for gene expression analysis. The reliance of TempO-LINC on the TempO-Seq technology makes it a fundamentally different and novel approach to single-cell genomics. Instead of reverse transcribing RNA, TempO-Seq uses detector oligos (DOs) to specifically hybridize to two immediately adjacent regions within an expressed transcript. The two DOs hybridized to each transcript are then ligated together forming a DNA product that can be amplified and sequenced, thereby enabling the specific and sensitive detection of gene expression. This approach has many advantages including the ability to either be targeted to small numbers of transcripts or highly multiplexed across the entire transcriptome, reduced read mapping and bioinformatics challenges, reduced sequencing costs and the ability to assay gene expression on fixed cells that are banked in the freezer.
APPROACH
TempO-LINC relies on the exact same DO pools that BioClavis currently sells for whole transcriptome analysis; however, once ligated within fixed cells, the DO product is barcoded through a combinatorial split-pool approach. Different rounds of splitting and pooling of the fixed single-cell suspensions enable unique barcodes to be generated for each cell. Following our proprietary split-pool barcoding step, samples are PCR amplified and purified without the need for template switch synthesis, fragmentation, or SPRI selection steps. Unlike droplet-based methods which ultimately have finite limits on microfluidic processing and sample volumes, TempO-LINC is a truly scalable approach that is limited only by the number of barcodes in each round and the number of rounds of barcoding.
TempO-LINC Workflow
High-Efficiency Cell Barcoding
Figure 1. TempO-LINC analysis was performed on a fixed single-cell suspension of human HEK 393 and mouse NIH 3T3 cells. High-efficiency cell barcoding; Cell calling is demonstrated in the knee plot showing barcodes ranked by read depth. A clear inflection point can be identified for bioinformatically identifying cells with a read depth greater than 1000 (shown in blue). Low doublet rate; TempO-LINC doublet rate was assessed as <0.5% for 12,000 cells analyzed. This is exceptionally low and almost identical to the theoretical clash rate of 0.45% for this experiment. Doublets are shown in red on the mixed species plot. Avg. genes detected per cell; TempO-LINC shows an excellent gene discovery rate that is competitive with other marketplace solutions. Greater than 5,000 human and mouse genes are detected at a depth of 50,000 reads per cell. Genes detected vs transcripts; The probe-based nature of TempO-LINC allows a greater number of genes to be detected for a given number of transcripts identified relative to competing platforms in human HEK 293 cells. The ability to ignore non-informative content (e.g. mitochondrial genes) and attenuate the signal of highly expressed genes is a key factor in this ability to resolve greater complexity of single-cell gene signatures.
UMAP Analysis
Figure 2. UMAP analysis showing results from a TempO-LINC customer study. A total of 48 human hepatocyte samples were treated with vehicle only or one of 7 different drug compounds. Over 74,000 cells were identified following TempO-LINC with an average detected gene rate of 4,837. Different compounds as well as different doses of compound are capable of driving unique gene expression signatures that are resolved with TempO-LINC.
PRODUCT SPECIFICATIONS
Full service offering on BioClavis Human and Mouse Whole Transcriptome gene panels with the ability to measure custom content