Question: What are the additional peaks in my Single Cell Gene Expression library?
Answer: After library construction, Single Cell Gene Expression libraries are run on the Bioanalyzer, Tapestation, or LabChip 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 within the typical size distribution for final Single Cell Gene Expression libraries, ~300-1000 bp. The library trace is representative of the biology of the sample and should be fine for sequencing.
If sharp peaks <200 bp are present, 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:
Inefficient SPRIselect cleanup of post cDNA amplification reaction and/or post-Sample Index PCR.
Recommendations for minor peaks below 200 bp
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 the percentage of adaptor dimers out of the total raw reads before sequencing can not be accurately quantified based on the trace alone.
*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.
Recommendations for more abundant peaks below 200 bp
If a substantial proportion of the library is <200 bp fragments, and total library yield is sufficient, it is recommended to perform an additional SPRIselect cleanup before sequencing to remove adaptor dimers. Note that approximately 40% of the material may be lost. If the total library yield is insufficient, we recommend redoing library preparation from left-over amplified cDNA.
Suggested protocol for removing peaks below 200 bp:
- Add nuclease-free water to bring the library volume to 100 ul.
- Repeat the SPRIselect cleanup in the "Post Sample Index PCR Double Sided Size Selection" step (this is step 3.6 in the Single Cell 3' v3.1 Dual Index User Guide and uses a double-sided SPRIselect ratio of 0.8X (left side ratio) and 0.6X (right side ratio).
- This SPRIselect cleanup can be performed on an individual library or a library pool (for libraries that will be sequenced together).
Please refer to the Single Cell 3' v3.1 Dual Index User Guide, for example, images of libraries with adaptor dimer peaks that were removed by performing an additional double-sided SPRIselect cleanup:
Note that instead of repeating the double-sided SPRIselect cleanup in step 3.6, it may also be feasible to perform a single-sided SPRIselect cleanup using a 0.8X or 1.0X SPRIselect ratio. Please refer to the SPRIselect manual for further guidance on performing single-sided SPRIselect cleanups and selecting an appropriate SPRIselect ratio based on the fragment size of the adaptor dimer peaks. A SPRIselect ratio of 1.0X may be sufficient for removing most adaptor dimer fragments, whereas a more stringent SPRIselect ratio of 0.8X may be necessary if the adaptor dimer fragments are larger (~200bp).
Protocol for single-sided 0.8X or 1.0X SPRIselect cleanup
- Add SPRIselect reagents to your library or library pool, at a ratio of 1.0X or 0.8X
- (eg. if you have 30 ul library, add 30 ul SPRIselect for 1.0X ratio, or 24 ul SPRIselect for 0.8X ratio).
- 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 and discard the ethanol
- Add 200 ul 80% ethanol to the pellet. Wait 30 sec. Remove the discard the ethanol
- Centrifuge briefly and place on magnet
- Remove and discard the remaining ethanol
- Remove tubes from the magnet. Add 30.5 ul Qiagen 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 (this is the cleaned-up library). Discard the bead pellet.
Products: Single Cell Gene Expression, Single Cell Immune Profiling