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 adaptor 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 adaptor dimers out of the total raw reads before sequencing.
*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.
If a substantial proportion of the library is <200 bp fragments, and total library yield is sufficient, it is recommended to perform an additional 1.0X SPRIselect cleanup prior to sequencing to remove adaptor dimers. Note that approximately 40% of material may be lost. If the total library yield is insufficient, we recommend redoing library preparation from left-over amplified cDNA.
Protocol for performing 1.0X SPRIselect cleanup to remove adaptor dimers:
- add an equal volume of SPRIselect reagent to your Gene Expression library (eg. add 30 ul of SPRIselect to 30 ul library)
- pipette mix 15x
- incubate 5 min at room temperature
- place on magnet until the solution clears
- remove and discard the supernatant
- add 200 ul 80% ethanol to the pellet. Wait 30 sec. remove the ethanol
- add 200 ul 80% ethanol to the pellet. Wait 30 sec. remove the ethanol
- centrifuge briefly and place on magnet
- remove remaining ethanol
- remove tubes from the magnet. Add 30.5 ul Buffer EB to the pellet. Pipette mix 15x.
- incubate 2 min at room temperature
- place the tube strip on the magnet until the solution clears
- transfer 30 ul to a new tube
Products: Single Cell Gene Expression, Single Cell Immune Profiling