SeqWell Innovations for Low-Cost Single-Cell RNA Sequencing

Advancements in single-cell RNA sequencing (scRNA-seq) have transformed our ability to study gene expression at an individual cell level. However, traditional methods remain expensive and technically complex, limiting accessibility. Lowering costs while maintaining accuracy is crucial for expanding scRNA-seq applications.

SeqWell has developed innovations that streamline the process, reducing expenses without compromising data quality. By improving microfabrication techniques, refining single-cell isolation, and utilizing efficient molecular barcoding strategies, SeqWell offers a scalable alternative to conventional methods.

Microfabrication And Chamber Design

SeqWell’s platform achieves efficiency and cost-effectiveness through advancements in microfabrication and chamber design. Traditional microfluidic systems often require labor-intensive fabrication techniques that increase costs and limit scalability. SeqWell addresses these challenges with a simplified, high-throughput microfabrication process that enhances reproducibility while reducing material expenses. Using soft lithography and photopolymerization, SeqWell produces microfluidic devices with precise geometries that optimize fluid dynamics, ensuring uniform cell capture and reagent distribution.

A key feature of SeqWell’s chamber design is its structured microwell array, which partitions individual cells efficiently. Unlike droplet-based systems that require continuous flow and precise emulsification, SeqWell’s static microwell approach minimizes mechanical stress on cells, preserving their integrity. Hydrophilic coatings promote even cell settling, reducing doublet formation and improving resolution. Chamber dimensions optimize cell capture efficiency and reagent diffusion, ensuring uniform molecular barcoding reactions.

Material selection is critical to performance. Optically transparent polymers, such as polydimethylsiloxane (PDMS) or cyclic olefin copolymers (COC), enable real-time imaging and quality control during cell loading. These materials exhibit low autofluorescence, essential for fluorescence-based quality assessments. Surface modifications, such as oxygen plasma treatment or polyethylene glycol (PEG) grafting, prevent nonspecific adhesion, reducing background noise in sequencing data.

Single-Cell Isolation Procedure

The precision of single-cell RNA sequencing depends on isolating individual cells without introducing bias or contamination. SeqWell’s microwell-based approach avoids challenges associated with droplet microfluidics, such as inconsistent encapsulation and shear stress-induced transcriptional artifacts. The static array of wells ensures each cell remains in a controlled microenvironment, minimizing external influences that could alter gene expression. This method enhances reproducibility and simplifies the workflow, making it accessible to laboratories without specialized microfluidic expertise.

Cell loading occurs through passive sedimentation, optimized to maximize single-cell occupancy while reducing doublet formation. Hydrophilic surface coatings promote even dispersion, preventing aggregation and ensuring uniform distribution. Computational modeling of fluid dynamics informs microwell design, balancing well dimensions with cell settling kinetics to achieve high capture efficiency. Studies show this approach consistently yields single-cell capture rates exceeding 80%, surpassing conventional microfluidic droplet methods.

Once cells are positioned, a controlled lysis step releases RNA while preserving spatial separation. SeqWell’s mild detergent-based lysis buffer minimizes RNA degradation while ensuring complete cytoplasmic release. Unlike harsher chemical or mechanical lysis strategies that can introduce fragmentation biases, this approach preserves transcript integrity for more accurate downstream analysis. The lysis reaction is carefully timed and monitored through real-time imaging to ensure uniform processing before molecular barcoding.

Bead-Based Molecular Barcodes

SeqWell’s bead-based molecular barcoding system enhances efficiency and accuracy by providing a scalable approach to transcript labeling. Unlike traditional microfluidic-based barcoding, which relies on encapsulating individual cells with molecular tags in oil droplets, SeqWell utilizes a solid-phase bead system that eliminates the need for complex fluidic control. This simplifies the workflow while maintaining high-throughput capabilities, making it an attractive alternative for researchers seeking to reduce costs without sacrificing data integrity.

Each bead is functionalized with oligonucleotide barcodes that uniquely tag transcripts from individual cells. These barcodes include a unique molecular identifier (UMI) and a cell-specific sequence, ensuring RNA molecules can be accurately traced back to their originating cell during data analysis. Beads are preloaded into microwells before cell capture, allowing direct hybridization of mRNA upon lysis. This minimizes crosstalk between samples, as each well remains physically isolated, preventing barcode contamination common in droplet-based methods.

Hybridization efficiency depends on bead surface chemistry and oligonucleotide density. SeqWell optimizes these parameters to maximize RNA capture while preventing steric hindrance, which can reduce efficiency. The beads feature a high-binding capacity polymer matrix that facilitates rapid and stable RNA attachment, ensuring even low-abundance transcripts are captured. Studies demonstrate this method achieves a high transcript recovery rate comparable to more expensive commercial platforms, making it a viable option for large-scale single-cell studies.

Library Construction

Once transcripts are uniquely barcoded, constructing a high-quality sequencing library is essential for accurate gene expression data. SeqWell employs an optimized reverse transcription process, converting mRNA molecules hybridized to barcoded oligonucleotides into complementary DNA (cDNA). Reaction conditions are carefully calibrated to enhance efficiency while minimizing bias, ensuring the full diversity of transcripts is captured. Enzymatic fidelity is critical, as errors introduced during cDNA synthesis can propagate through amplification, distorting quantification. Using a high-processivity reverse transcriptase with low error rates, SeqWell maximizes transcript accuracy.

Following reverse transcription, excess primers and unbound oligonucleotides are removed to prevent spurious amplification. A bead-based purification system selectively retains cDNA while eliminating unwanted reaction byproducts, improving downstream amplification efficiency. The cDNA molecules undergo a pre-amplification step using a limited-cycle PCR protocol that maintains transcript proportionality. Over-amplification can introduce GC-content bias, skewing gene expression estimates; SeqWell mitigates this risk by empirically determining the optimal cycle number for each experiment. This ensures both high- and low-abundance transcripts are equally represented in the final library.