INTRODUCTION
BioSpyder is offering a proprietary high-throughput single-cell genomics platform, TempO-LINC (Ligation INdexed Cells) . This product relies on the foundational TempO-Seq technology approach developed by BioSpyder 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.
TEMPO-LINC WORKFLOW
APPROACH
TempO-LINC relies on the exact same DO pools that BioSpyder currently sells for whole transcriptome analysis; however, once ligated within fixed cells, the DO product is barcoded through multi-step barcoding which enables unique identifiers to be generated for each cell. Following our proprietary 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.
SCALABLE AND SENSITIVE SINGLE-CELL TRANSCRIPTOME PROFILING
Figure 1. TempO-LINC analysis was performed on a fixed single-cell suspension of human HEK 393 and mouse NIH 3T3 cells. A) 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). B) 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. C) 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. D) 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.
TEMPO-LINC UNCOVERS NOVEL INSIGHT INTO CELL SUBTYPES
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.