Question: Why do my V(D)J enrichment and V(D)J library traces look different from the user guide?
Answer: It’s common to see variabilities in the V(D)J enrichment and library traces. The appearance of the V(D)J enrichment and library traces is very sample-type specific, and some variation is normal. The appearance is based on many factors, including species (human vs. mouse), the number of T or B cells in the sample, how many V(D)J transcripts are expressed by those cells, which instrument the trace was run on (Tapestation vs. BioAnalyzer). In both the V(D)J enrichment and the V(D)J library traces, the critical point to observe is that there is a sharp peak between 500 and 700 bp--this peak contains your V(D)J transcripts.
Additional peaks at between 2kb and 10kb
The HMW products in the post-target enrichment QC are non-specific background products that are generated due to performing two enrichment reactions for TCRs from the total transcriptome. For the samples we have tested in-house, the high molecular weight product does not affect application performance as long as the library is sequenced to our minimum recommended depth of 5000 read-pairs per cell.
Representative Enriched TCR Trace
These peaks are usually more prevalent in TCR enriched samples than Ig enriched samples because of the lower TRA/TRB expression compared to IGH/IGK/IGL. They are also more prevalent when the percentage of T/B cells is low.
Low V(D)J enrichment yield
When experiencing low V(D)J enrichment yield, you may consider increasing the amount of cDNA (e.g., 5ul instead of 2ul) used for V(D)J amplification without adjusting the other volumes and/or increasing the enrichment PCRs by 1-2 cycles. Please note, this is an untested workflow modification by 10x Genomics and can be dependent on the sample type.
Prominent, large peak <500 bp in V(D)J library trace
In some cases, a prominent, large peak below 500 bp can be present in the V(D)J library trace. This could be related to a low frequency of T/B cells or the sample type (e.g., whether it was enriched T cells/B cells or a total cell population).
This large peak contains shorter V(D)J fragments that will still map to any V(D)J gene and these shorter fragments are used for the contig assembly as well.
If there is a distinguished peak between 500-700 bp in the V(D)J library trace, and the cDNA traces and V(D)J enrichment traces look as expected, it should be low risk to sequence these samples.
Products: Single Cell Immune Profiling