Isolated RNA, as low as 20 ng, is converted to cDNA. This is followed by the library construction step, in which IL-N7 adapters, molecular barcodes, and sample indexes are incorporated into DNA fragments generated in the previous step. Library fragments now serve as templates for target enrichment using single primer extension. In this step, targets are enriched using a single gene-specific primer and a universal forward primer. The final step is library amplification and sample indexing (for dual indexing) using the IL-S5 sample index primer and a universal primer.
Due to PCR duplicates generated in amplification steps, all fusion library fragments look exactly the same, and there is no way to tell whether a specific fusion library fragment is a unique fragment or a duplicate of another fragment. With molecular barcodes, since each unique fusion transcript is barcoded before any amplification takes place, unique library fragments are identified by their unique barcodes, and PCR duplicates carrying the same barcode are removed, thereby increasing the sensitivity of the panel.
The TCR richness of each receptor per sample is revealed.
V-J usage heatmaps for a single sample are shown. Jurkat RNA was spiked into normal human PBMC RNA at a dilution of 1:10,000 (800 Jurkat cells in 8,000,000 PBMCs).
The heatmaps allow for easy identification of enriched clonotypes across the sample. Here, we see the major clonotype of the Jurkat cell, as well as the diversity of the PBMC background.