PIPseq CRISPR Mode

DRAGEN also supports processing samples from Illumina's CRISPR Single Cell kits using PIPseq technology. Setting --scrna-enable-pipseq-crispr-mode to true activates this mode.

Activating PIPseq CRISPR mode automatically configures DRAGEN for processing feature reads containing the CRISPR guide RNA (gRNA) sequences. This includes handling offsets in the cell-barcode position for the gRNA reads, transforming the gRNA cell-barcodes to match the gene expression ones, utilizing the "hook and grab" approach for identifying the gRNA reads, and counting the gRNA reads (disregarding BIs and IMIs). Both gene expression and gRNA reads are processed in the same single cell workflow, so extra steps are added to identify the hook sequence of gRNA reads. Note: unmapped reads do not contribute to gene expression read counts but are still included in gRNA counts if they match the hook sequence.

The “hook and grab” method is a targeted approach for identifying CRISPR perturbation reads. It leverages a conserved sequence within the guide RNA structural region as a “hook” to locate the guide RNA and then “grabs” the specific guide by mapping it to a database of known sequences based on their displacement from the hook.