Question: How do I design a custom targeted assay for Single Cell V(D)J?
Answer: It is possible to design custom enrichment primers to amplify targets of interest during the Single Cell V(D)J workflow, in place of the Human or Mouse T Cell Mix 1 & 2 or Human or Mouse B Cell Mix 1 & 2 which amplify the T-cell Receptor (TCR) or Immunoglobulin (Ig) transcripts, respectively. Custom assays are not supported by 10x Genomics and will be the responsibility of the user to provide optimization for their particular assays.
To develop a targeted assay, we recommend consulting the following Technical Note, which details the Single Cell VDJ assay scheme, primer sequences, as well as enrichment primer concentrations, for guidance:
Note that TCR and Ig genes are typically expressed at low levels and therefore the PCR conditions and cycling may also need to be optimized depending on the expression level of the mRNA target(s). Genes with low levels of expression are not good candidates for targeted sequencing since they are unlikely to be detected at a high enough level to give adequate representation across cells in the sample.
A few additional notes:
- The number of Target Enrichment cycles may need to be increased by 1-2 cycles, particularly if there is a low relative abundance of the transcript of interest.
- The number of SI-PCR cycles will likely need to be increased by 1-2 cycles since the total initial mass will be significantly less.
- Depending on the length of the transcript(s) of interest, the parameters for fragmentation may need to be optimized to prevent excessive fragmentation. Fragmentation has been optimized for cDNA originating from full-length mammalian transcriptomes and thus fragmentation from a selected group of transcripts may require protocol adjustments. The length of the fragmentation reaction (2 minutes) can be increased or decreased accordingly.