Better, cheaper, easier, HyDrop
New open-source single-cell sequencing platform outperforms its predecessors
March 24, 2022
25 March 2022
A Leuven research team led by Stein Aerts and the single-cell experts at the VIB-KU Leuven Center for Brain & Disease Research has developed HyDrop: an optimized open-source protocol using dissolvable hydrogel beads. HyDrop reduces both the cost and labor intensity of existing open-source single-cell sequencing methods and offers improved sensitivity. The new method, published in eLife, will accelerate the implementation of single-cell analysis in a variety of research fields.
Every drop counts
Thanks to the advent of single-cell technology, researchers can study biological tissues at unprecedented resolution.
To be able to study single cells individually, researchers divide them over tiny droplets. Individual cells are rapidly encapsulated into a nanoliter droplet together with a barcoded gel bead. These barcodes are important to index each individual cell’s genetic material.
The content of thousands of single cells can then be pooled and processed for next-generation sequencing, allowing for high throughput and low reagent consumption. In this way, researchers across the globe have been able to profile the gene expression of tens of thousands of single cells at an affordable cost.
Open-source vs commercial
One of those researchers is Suresh Poovathingal, microfluidics expert at VIB and KU Leuven. “We have witnessed a true revolution in the field of single-cell genomics over these last few years and thanks to droplet microfluidics (and similar platforms), there are several commercial and open-source techniques available for researchers looking to do high-throughput single-cell sequencing.”
Some techniques use hard resin beads to carry uniquely barcoded primers into the droplets. To prevent obstruction, these resin beads are often loaded at dilute concentrations, but this also means that many droplets remain empty, leading to a cell capture rate as low as 2% and quite some wasted reagents. Deformable hydrogel beads, on the other hand, can be stacked and loaded into droplets at a fixed rate without risk of microfluidic failure, increasing the cell capture rate to 50% or higher.
Prof. Stein Aerts (VIB-KU Leuven): “Several commercial solutions have been developed and successfully applied to generate hundreds of thousands - and recently millions - of single-cell transcriptomes at high sensitivity. They are not cheap though, and their “closed box” approach limits custom protocol development.”
“Existing open-source droplet microfluidic protocols are cheaper,” says Poovathingal, “but also have their downsides: they are quite cumbersome and their sensitivity is limited.”
The best of both worlds
In order to increase the sensitivity and user-friendliness of open-source microfluidic protocols, and to provide a more flexible and open platform, Aerts and Poovathingal set out to develop HyDrop, a new hydrogel-based droplet microfluidic method for high-throughput single-cell sequencing.
“We adapted existing bead barcoding protocols to generate more uniformly barcoded beads,” explains Florian De Rop, PhD student in the Aerts lab. “Next, we modified the hydrogel bead production process to generate dissolvable beads, improving barcoded primer release and diffusion inside the droplet.”
The team also optimized the reverse transcription strategy (translating RNA to DNA) so it could be applied inside the cell/bead emulsion. The assay’s sensitivity was optimized by testing several different preparation strategies.
“The combination of all these adaptations resulted in a significantly increased sensitivity at no additional cost, and at the same time a more user-friendly workflow,” says De Rop.
Poovathingal: “With our new approach we were able to assess genome accessibility through single-cell sequencing with an open-source method in droplets using hydrogel beads. A first!”
Cheaper, easier, and more sensitive
The team applied HyDrop to generate thousands of mouse, human, and fruit fly single-cell gene expression and chromatin accessibility profiles, demonstrating the protocol’s applicability to a variety of different biological samples. The results have been published in eLife.
“HyDrop experiments on mouse and fly tissues recapitulated the cellular heterogeneity of these complex samples and results were in line with reference datasets,” says Aerts. “HyDrop also outperformed its open-source predecessors both in terms of sensitivity and user-friendliness, and at a significantly lower cost compared to commercial droplet-microfluidic alternatives.”
The researchers are convinced that HyDrop has big potential for the research field: “The reduced cost and labor will accelerate the scaling of large-scale cell atlasing efforts and bring the benefits of single-cell sequencing to smaller projects,” says Poovathingal, who continues to look for new ways to further improve HyDrops performance and sensitivity.
“Our dissolvable bead synthesis and barcoding toolkit could also be exploited to produce more complex beads, incorporating multiple capture sequences. This could be helpful for the implementation of single-cell (multi-)omics assays to capture not only genetic material from single cells but also intracellular proteins, for example. ”