10X Genomics recently launched new versions of their flagship 3′ and 5′ Gene Expression assays integrating an approach they call “On-Chip Multiplexing” or OCM. The OCM assays feature a relatively very low per sample reagent cost compared to other 10X products, and these versions of the assays have some distinct advantages over other multiplexing options, but they also have their own unique limitations. The purpose of this post is to focus primarily on those strengths and limitations – if you are interested in more technical detail regarding how OCM is different from the standard 3′ and 5′ assays, this information is discussed in more depth in a longer version of this post here.
In the OCM assays, a special variant of 10X’s microfluidic chips is used to feed up to four individual sample/reagent input blocks into a single GEM formation/collection well. This is enabled by the use of a known, non-overlapping set of cell-identifying barcodes (which come from the Gel Bead reagent shown in the below diagram) used for each initial sample. Because we know that a given sample was loaded in a lane with a specific known set of gel beads with a unique subset of cell barcodes, we can link any cells in the data back to their original source sample even though they have all been pooled together into a single GEM reaction for the purposes of constructing a single cDNA library containing all four samples.
Strengths and limitations of the OCM assay
The most substantial benefit to using the OCM assay is the price of the reagents. As of February 2025, the list price of a 16-reaction/sample kit for the standard, non-OCM 3′ Gene Expression assay is approximately $23,700. A 16-sample kit for the OCM version of the assay has a list price of approximately $9,000. Outside of this cost reduction in the main library preparation kit, additional “per library” consumable costs, including materials needed to QC the library at the cDNA stage and the final library stage, will also be lower since we are working with 1/4th as many libraries.
The other significant strength of this assay as a multiplexing option is that it avoids some of the more challenging aspects of using a cell labeling-based approach like antibody hashing. Namely, you do not need the relatively high numbers of starting cells (1M or more) that BioLegend recommends as input for their staining protocols, and since the cells do not need to be put through the additional extensive hands-on processing required to label them, we can get them loaded into the assay in a relatively fresh state and minimize the sort of cell health impacts that can potentially arise as the time between the initial collection of the cells and their capture in the 10X assay increases.
On the other hand, the OCM has a few notable limitations. These will be discussed in more detail below, but in short:
- The number of cells per sample we are able to capture is much lower – 5,000 cells per sample in OCM versus 20,000 cells per sample in the standard 3′ or 5′ assays.
- There is somewhat reduced flexibility in terms of the number of samples that can (or should) be run at once, especially compared to projects using the standard 3′ assay in our core.
- The OCM assay is incompatible with “sort & load” experiments where a very rare cell population (~10,000 cells or fewer) is sorted into a small volume of buffer and loaded directly into the 10X assay.
The most significant limitation of the OCM assay is a much lower cap on the number of cells that can be captured per sample. In the standard/non-OCM version of the assay, each of the eight lanes on the chip has a recommended maximum cap of 20,000 captured cells per sample. In the OCM assay, the maximum recommended capture target is only 5,000 cells per sample (meaning that the total combined GEM reaction is still capped at 20,000 captured cells). 10X Genomics sets this cap based on the “multiplet rate”, which is the proportion of GEM units that are expected to contain more than one cell. This multiplet rate is much higher in the OCM assay – putting in enough cells to target 5,000 cells per sample in this assay will result in the same ~8% multiplet rate that is seen when targeting 20,000 cells in the standard version. We can get around this limitation to some extent by loading samples multiple times – e.g. instead of loading four samples and getting 5,000 cells for each sample, we load two samples in two lanes each in order to capture 10,000 total cells from that sample – but this rapidly erodes the per sample cost savings for the assay and may still be undesirable if you are trying to capture a relatively rare cell population that would significantly benefit from being able to get a full 20,000 cells per sample.
In terms of the flexibility for running different sample numbers – for the standard 3′ Gene Expression assay, the Gene Expression Center stocks the 16 reaction kits. This allows our client base to run however many samples they wish to run for their experiment without needing to commit to larger reagent costs. If someone only wants to run one or two samples, they will only pay for one or two units of the library preparation reagents instead of needing to buy (at minimum) a four-sample kit. For the OCM kits, unless they become popular enough that we can be sure we won’t have reagents expiring unused, we would need to order a 16-sample kit specifically for your project and bill you for the full cost of that kit, even if you aren’t necessarily interested in running 16 samples (or even eight samples with double-loading). Additionally, the nature of the multiplexing in the kit is essentially “use it or lose it” since gel beads must still be used in a lane even if that lane receives only a mock sample (PBS) in the sample well. If you want to run 16 total samples, but are limited to only being able to prepare two samples at once (e.g. due to a lengthy sample prep or required sort), we would need to order multiple kits.
Lastly, regarding projects involving the sorting of a very rare cell population. This approach is possible in the standard version of the 3′ assay because the maximum volume of sample that can be put in to each lane is around 37 uL. This volume is usually pretty close to the volume of sample that we will have when roughly 10,000 cells are sorted directly into a strip tube with a relatively small nozzle (to minimize the buffer volume carried through per cell), including a 10 uL “buffer cushion” placed into the tube at the start to catch the sorted cells. In the OCM assay, since we are essentially breaking each GEM reaction into four separate inputs, the maximum volume we can load per sample is only 9.6 uL. This volume is far too small to be able to run these sorts of samples without trying a centrifugation step, which itself is likely to be extremely risky with so few cells.