Question: What are the additional peaks in my final library QC?
Answer: After library construction, the 10x Single Cell 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.
If an abundant cell-type specific transcript is present, additional peaks may be present on the Bioanalyzer or TapeStation trace within the typical size distribution for final libraries, ~300-1000 bp. The library trace is representative of the biology of the sample and should be fine for sequencing.
If there are sharp peaks <200 bp, this may indicate carryover adaptor/primer dimers in the final library and may affect sequencing results*.
If you observe small peaks (<200 bp) in the post-library construction trace:
Causes:
Inefficient SPRIselect cleanup of post cDNA amplification reaction and/or post Sample Index PCR.
Recommendations:
If the integrated area beneath the 200 bp peak is much smaller than the overall integrated area beneath the desired library fragments (~300 bp to 1 kb), and the user can tolerate the presence of some adapter dimers (which will reduce the amount of sequencing information*), the library can be sequenced after KAPA quantification. Please note that based on the trace alone, one will not be able to accurately quantify the percentage of adapter dimers out of the total raw reads before sequencing.
If a substantial proportion of the library is <200 bp fragments, and total library yield is sufficient, it is possible to perform a second SPRIselect cleanup prior to sequencing. Please follow Step 3.6 of the User Guide (Post Sample Index PCR Double Sided Size Selection – SPRIselect) for specific instructions. Note: approximately 40% of material may be lost when repeating the cleanup.
*SAV metrics can be affected when as little as 1% of the sequencing library is adaptor dimers. Specifically, the "% Base per cycle" may have an uneven proportion of bases representing the adaptor sequence. This may have downstream effects on quality scores, in addition to losing adaptor reads as wasted data.
Products: Single Cell 3', VDJ