Why isn’t the recommended pooling ratio for VDJ libraries proportionate to the recommended reads/cell?

TCR and BCR libraries have longer fragments than targeted mRNA libraries. Longer fragments are more likely to be outcompeted by smaller fragments during clustering, which results in less data output than expected. Our pooling ratio recommendation has been optimized and validated to obtain the recommended data output ratio for each library.

Is the targeted mRNA library necessary for the VDJ CDR3 assay? Can I only sequence BCR and TCR libraries?

At minimum, there are three libraries for VDJ CDR3 analysis. Currently, the pipeline requires mRNA reads for cell calling. If an mRNA library is not added, the bioinformatics pipeline will crash at the cell calling step.

Can I use panels other than the immune response panel or custom panel with the VDJ protocol?

While the VDJ CDR3 protocol can technically work with any targeted panel, the immune response panel has genes specific for immune cells to aid immune cell calling, a critical feature for VDJ output. Additionally, to work effectively with our CDR3 VDJ assay, custom panels should include immune response panel markers.

Can I pool the BD^® AbSeq Library with VDJ libraries to sequence them together?

We cannot guarantee an accurate data output ratio when combining the VDJ and AbSeq libraries. We recommend sequencing AbSeq libraries separately in a PE75 cycle sequencing setup as the small AbSeq fragments will outcompete VDJ libraries. If it is necessary to run the AbSeq libraries with the VDJ library, we recommend using a unique indexing reverse primer for the AbSeq libraries so that the AbSeq reads can be trimmed before running the primary analysis pipeline.