Droplet microfluidics has made a large impact in many different applications, ranging from the analysis of DNA variants to secretory biomolecules and diverse cell types. However, to enrich individual variants of interest, researchers face challenges with high-throughput screening and isolation of these emulsion droplets. Integrated strategies that combine the sensitivity and precision of cell sorting with downstream single-cell multiomic analysis are now being used to address these challenges.
In this webinar, Dr. Marie Mikkelsen, Co-founder and CTO of Samplix, and Kara Brower, Ph.D. Candidate at Stanford University, discuss how gel-encapsulated microdroplets can be analyzed by high-throughput droplet sorting for genomic, proteomic, and metabolomic profiling for a diverse range of applications.
Q: Can Xdrop technology be used to resolve large genomes, such as those of plant cells, and how does this compare to the PacBio approach?
KB: PacBio sequencing is good for whole-genome sequencing, whereas the Xdrop approach is mainly used for extracting DNA and then sequencing. This means the Xdrop is a perfect match for the PacBio. What we do is extract the DNA of the region that we are interested in and then load the information onto the PacBio. In doing this, you decrease the complexity of your sample by a thousandfold or more.
Q: What is the multiplet rate of technology developed by your lab compared to other methods such as Drop-Seq and inDrop?
MM: We recently released a new publication on this. We have shown that we have adjusted our multiplet rate to be less than 2.5%, whereas DropSeq and inDrop, as well as other commercial technologies such as 10x, typically have rates between 4% and 8%. You can find out more information about this in our new publication on bioRxiv.
Q: How does the stability of double emulsions compare to that of single emulsions? And can the droplets be incubated for more than 24 hours?
MM: We have found that the double emulsions are more stable than the single emulsions, and these can be incubated for 24 hours at 30 to 37 degrees.
Q: Do particle doublets or multiplets occur, and are they at risk of clogging the system?
KB: For large particles, yes, some doublets do occur. However, we have observed doublet rates in our D-FACTS paper to be typically less than 5%. However, it depends on the particle size. We have created a lot of different instrument parameters, such as using a large instrument nozzle, as well as spacing out the double emulsions using pluronic loading fluids. This has really helped to get a nice singlet rate and nice event rate during operation of either an Aria class order or a Sony class order. We have found that the Sony does very well with metering the droplets out very precisely for a later low abort rate during single-celled purity sort.
Q: Have you grown microorganisms inside of the double emulsions? If yes, how does this scatter signal look, and is it possible to distinguish empty and occupied drops based on this?
KB: We've grown yeast as well as many different types of bacteria, including very rare environmental species with one of our collaborators. We have had no problems growing them in the double emulsion. Some have even survived for up to a week. It really depends on how big your colony grows, and whether the colony will burst the double emulsion structure. We do get a more reliable signal from larger particle loading, such as mammalian cells. This is, again, in our recent publication, but we do have more forthcoming work that is focused on looking at bacterial colony growth within the double emulsion. Typically, beyond 10 to 50 cells you can see and sort those cells as well, though the single-cell level unlicensed is quite difficult.
Q: Is the droplet generation device, as well as the chips, available commercially? Does this always generate double emulsions, and can the droplet size be controlled?
MM: The Xdrop instrument and the single-use cartridges are commercially available. You can generate either small double emulsions or, using a different cartridge, you can make larger single emulsions. In our instrument, we cannot control the droplet style, but we can control the droplet size.
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