Question: How do I diagnose and report a clog failure with GEM-X assays?
Answer: When running GEM-X Chips, a clog may occur. A clog is evidenced by recovering an excess of Partitioning Oil in the recovered emulsions and may have <100 µl GEMs from the recovery well (see image below). Best practice is for recovered emulsions to be QC’d in pipette tips following emulsion retrieval (see figures below) and GEM-X Chip to be inspected for irregularities in remaining volumes across lanes. If other samples are run in parallel, the clogged sample wells may have uneven volumes of remaining reagents compared to other sample wells.
Causes:
- Clogs are generally caused by suboptimal sample preparations, non-sterile work environments, clumping of Gel Beads and/or slow chip loading.
Distinguishing between types of clogs:
- Sample clogs are the result of suboptimal sample preparation of cell/nuclei suspensions whereby debris or clumps impede the flow through the chip. They may result in a decreased rate of consumption of the sample with the recovered emulsion that is <100 µl and contains excess Partitioning Oil.
- Gel Bead clogs are caused by improper handling of Gel Beads. A clog in the Gel Bead line causes Gel Beads to flow slower, leaving an excess of Gel Beads behind. This may result in recovered emulsion that is <100 µl and contains excess Partitioning Oil. Gel Bead clogs are expected to very rarely occur.
If a clog occurs near the end of the GEM-X Chip run, the recovered emulsion volume may be ~100 µl but the emulsion is still expected to contain an excess of Partitioning Oil. Clogs could theoretically present themselves as recovered emulsions with <100 µl volume but no presence of excess Partitioning Oil. Clogs not containing excess Partitioning Oil have not been observed in-house with GEM-X Chips but we expect that the most likely root cause of this would also lead to a wetting failure (See: How do I diagnose and report a wetting failure with GEM-X assays?).
Recommendations:
- To minimize the risk of microfluidic clogs, we recommend storing and loading chips in an area free of dust and debris. For GEM-X Chips (where secondary holders are open while loading), a pipette tip lid may be placed over the chip itself and does not need to fully cover the opened portion with the gasket attached. Other materials other than pipette tip lids have not been tested, may introduce unwanted debris, and are not recommended.
- We strongly recommend rerunning the sample if a clog occurs during GEM generation. However, if it is a precious sample in which additional cell material cannot be obtained and cells would be lost otherwise, it may be possible to continue with the protocol and generate cDNA, albeit at lower levels, and with the knowledge that the number of recovered cells will be lower. Specifically, if the clog occurred near the end of the run, most cells will have been partitioned into GEMs. Similarly, if a large number of cells have been run, this may also increase the likelihood of generating sufficient amounts of cDNA.
Next steps:
- As part of our warranty policy, clog and wetting failures that have been properly documented are reimbursed with replacement reagents and chips.
- Please send (1) the lot numbers of the Gel Beads and chips that were used when the failure occurred, (2) date of the failure, (3) a picture of the failed emulsions in the pipette tips/strip tubes and the chip (see this article: How should I take photographs to document clogs and wetting failures?), and (4) a recent purchase order and a contact phone number to support@10xgenomics.com.
- Clogs are not eligible for replacement if expired reagents or chips were used.
- Clogs must be reported within 30 days of the expiration date, as stated in our Terms and Conditions.
- Clogs associated with an instrument error are only eligible for replacement if the instrument is under an active Warranty or Assurance Plan at the time of the failure.
Best practices:
- Make sure Gel Beads are completely thawed and vortex prior processing.
- Handle Gel Beads in a sterile environment.
- Store chips in an area free of dust.
- Do not use chips or reagents beyond their expiration date.
- After removing the chip from the sealed bag, use it within 24 hours.
- Dissociate cell clumps and aggregates and remove cell debris using cell strainers/filters; visually inspect single cell suspension before loading onto the chip.
- Do not overload chip with excess cells.
- Minimize the volume of single cell suspension to reduce likelihood of debris carryover to sample line (increase cell concentration).
The appearance of emulsions from the recovery wells of the GEM-X Chip in pipette tips. The sample on the left is a successfully generated emulsion. The sample on the right has reduced emulsion volume and an excess of Partitioning Oil (clear), indicating a clog. In some reagent clog cases, there is excess Partitioning Oil (and no air) in the pipette tip.
Please note: lower emulsion volume recovered (<100 µl) without excess Partitioning Oil in the pipette tip may be due to pipetting errors during GEM-X Chip loading or recovering emulsions. As these emulsions are not clogs, they would not be eligible for warranty replacements. We would recommend checking that a well-calibrated multichannel pipette is used for chip loading and closely follow User Guide instructions for emulsion recovery.
Products: Single Cell Gene Expression, Single Cell Immune Profiling