Question: What are the additional peaks in my Cell Surface Protein library?
Answer: After library construction, the Cell Surface Protein library is run on either the Agilent Bioanalyzer High Sensitivity chip or Agilent TapeStation High Sensitivity D1000 Screen Tape at a 1:10 dilution for qualitative analysis. The traces should resemble the overall shape of the electropherograms shown in the User Guide. We expect to observe a narrow peak at ~220 bp.
There may be additional peaks present in the QC trace on rare occasions. We've observed the following examples.
Secondary peak present between 300 and 400 bp:
This peak is caused by 'daisy chains,' which result from over-amplifying the library leading to depletion of available primers and/or dNTPs. This will then result in the self-priming of PCR products by their P5 and/or P7 adaptors. Products resulting from this self-priming consist of essentially Cell Surface Protein library sequences attached to one another in 1 long oligo that is twice as long as the original library oligo; these products appear as peaks at approximately 400 bp. To reduce the presence of this secondary peak, you can reduce the number of SI-PCR cycles during the library construction step from 10 to 8.
Secondary peak present between 140 and 160 bp:
This peak is caused by off-target amplification and can sometimes arise when there is a relatively low presence of Cell Surface Protein signal (this can result when the cell surface protein expression is low, the total number of cells loaded is low, the antibody panel is small, or some combination of the three). This low level of Cell Surface Protein oligo signal can increase the chances of off-target amplification. Libraries with a minor off-target peak are fine to sequence; there may be a decrease in the 'Fraction Antibody Reads' metric but the Cell Surface Protein signal will still be present and consist of the majority of the reads.
Recommendations for additional peaks between 140 and 160 bp:
If the off-target peak appears larger than in the above image, contact firstname.lastname@example.org for guidance. There are a few troubleshooting avenues to consider.
While the following workflow modifications are not officially tested or supported by 10x, they have been used by customers to troubleshoot Cell Surface Protein libraries with low yields or if the Cell Surface Protein libraries contain off-target products, including primer carryover.
- Option 1: Repeat Sample Index PCR during Cell Surface Protein Library Construction- with the following workflow modifications:
- Use 10ul of DNA sample from the Transferred Supernatant Cleanup (Step 2.3B-ixv) instead of 5ul of the DNA sample if using standard assays
- Increase the total number of SI-PCR cycles by two cycles
- Option 2: Reamplify the final Cell Surface Protein library to enrich for the specific product:
- Do an additional 2-3 cycles amplification using P5/P7 primers (purchased separately from IDT)
- Repeat the 1.2X SPRIselect cleanup in the “Post Sample Index PCR Size Selection” step
(Option 1 seems to be generally preferred to resolve these off-target peaks.)
Recommendation for additional peaks < 100 bp:
It is recommended to perform an additional 1.0X SPRIselect cleanup before sequencing to remove carryover adapter/primer dimers.
Note on additional peaks greater than 1 kb.
In some cases, higher molecular weight peaks (>1 kb) may be present. These peaks are not problematic and do not negatively affect sequencing or assay performance.
Products: Single Cell Gene Expression, Single Cell Immune Profiling