Question: How can I optimize V(D)J primer design for my non-human/non-mouse species of interest?
Answer: In order to optimize the V(D)J primer design for non-human/non-mouse species of interest, you must first identify the transcripts that will be amplified, and collect a list of the C-gene sequences for these transcripts. Next, go through the transcripts and determine how many primer sequences would be needed to amplify all these transcripts. A good rule of thumb is that you will want to start with one outer and one inner primer for each isotype.
For the outer reverse primer, we recommend targeting a section about 200 - 300 bp away from the 5' end of the constant region. For the inner reverse primer, we recommend targeting a section between 50 - 200 bp away from the 5' end of the constant region. Ideally, you want the inner primer closer to the 5’ end of the C-gene in order to not waste too much sequencing data on the C-gene. Follow best practices for primer design, including limiting the % of GC content to 40 - 60%, and making sure that the melting temperatures of all the primers in a pool are within a similar range. Please note that the forward primer sequences can be found in the Appendix of the user guides and that the forward primer sequences are distinct between 5’ v1/v1.1 and 5’ v2. The forward primer for 5’ v2 was shortened to accommodate dual-indexing. The length of the forward primer will have an impact on the melting temperature of the pool.
Once you have a few viable options for your primers, search for each primer in your genome of interest. We recommend selecting a primer that has minimal interaction with other regions of the genome.
For QC of the primers prior to running your 10x experiment, you can design synthetic DNA pools which contain all your sequences of interest, and use your primers to enrich these pools and create a sequenceable library. This will allow you to verify that no important sequences of interest were missed during primer design. It will also allow you to determine if any primer concentrations need to be adjusted down or up if any specific sequences are unevenly represented in the pool.
We recommend a starting final primer concentration of between 0.25 uM and 1 uM. This may need to be adjusted up or down depending on the particulars of your sample type and species of interest to achieve balanced sequence enrichment.
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