Comparing Single-Cell RNA Sequencing Options

In only a few years, single-cell RNA sequencing (scRNAseq) has become more accessible to researchers who want to examine gene expression at the single-cell level—and the methods continue to evolve. Whether assessing the heterogeneity within a cell population, identifying cell types, or mapping single cells within tissues, there isn’t a one-size-fits-all method. Generally, most scRNAseq methods begin with isolating single cells and tagging their mRNAs with cell-specific barcodes or labels. Reverse transcription and library preparation then follow, prior to next-generation sequencing (NGS). Through subsequent analysis of sequencing data, the number of RNA transcripts in each cell can be counted. Here’s a rundown of the main scRNAseq methods available today, loosely organized by throughput—but the evolution continues, so stay tuned for new options.

Lower thoughput methods

Lower throughput methods might have a higher “per cell” cost compared to high-throughput methods, but depending on how many cells you need to sequence, this could beget savings. When estimating overall cost, make sure to include any instrumentation and consumables required by the platform, as this varies widely.

Standard BioTools Fluidigm C1™

In the C1™ system, the process of cell isolation, lysis, and library prep is automated within microfluidic channels and individual reaction chambers of a microfluidic chip. It can process hundreds of cells at a time, and is often used for screening with specific markers.

Takara Bio ICELL8 Single Cell System

Takara Bio’s ICELL8 Single Cell System uses nanowells to isolate individual cells for library prep prior to sequencing. As an open platform, its flexibility allows for a range of cell sizes and reagent solutions, and can sequence up to hundreds of cells per run.

Bio-Rad ddSEQ™ Single-Cell Isolator & Single-Cell 3’ RNA-Seq Kit

Bio-Rad’s ddSEQ™ Single-Cell Isolator isolates single cells in sub-nanoliter droplets containing barcoded beads. Cell lysis occurs within droplets, where RNA is labeled prior to library prep. This workflow requires the ddSEQ Single-Cell Isolator instrument and the Single-Cell 3’ RNA-Seq Kit; throughput is in the range of hundreds to thousands of cells per sample.

Illumina PIPseq

Illumina’s recent acquisition of Fluent Biosciences has made their PIPseq™ chemistry available in Illumina Single Cell 3′ RNA kits, with no need for special instrumentation or microfluidics. Instead, reactions are performed in tubes, vortexing cells and barcoded beads with an oil phase. This creates water-in-oil droplets, which each contain a single bead and cell.

Illumina Single Cell is flexible and scalable across many types of projects, supporting a wide throughput range of 200 cells to 1 million cells per reaction. “It enables single-cell analysis at all scales and often detects rare cell types, especially large, fragile, or sticky cells that are missed by microfluidic methods,” says Joel Fellis, Vice President of Product Management at Illumina.

The flexibility of the Illumina single-cell platform is convenient for moving from pilot studies to larger-scale projects. “Whether profiling complex tissues with many cell types or performing deep characterization of rare cells (e.g., circulating tumor cells), the combination of scalability, workflow flexibility, and streamlined analysis supports comprehensive, high-quality single-cell studies,” says Fellis. The Illumina single-cell solution includes DRAGEN secondary analysis and full integration into Illumina Connected Multiomics for deeper tertiary analysis and visualization.

Higher throughput methods

High-throughput scRNAseq is ideal for discovering rare cell types, enumerating cell atlases, characterizing cells and tissues, and may sequence thousands to millions of cells per run depending on the platform.

10x Genomics Chromium

A large supplier of sequencing kits, 10x Genomics offers the Chromium platform for scRNAseq, in which cells are isolated along with barcoded gel beads in individual oil droplets. RNA released from each cell is captured by barcodes and used to create sequencing libraries. This method requires their Chromium X or Chromium Controller instruments, and the HT kit can sequence over 2 million cells per run depending on the configuration. Scale Biosciences, which developed a split-pool method for sequencing millions of cells without special instrumentation using their Quantum Barcoding™, was recently acquired by 10x Genomics and will be integrated into their Chromium platform.

BD Biosciences Rhapsody

The BD Rhapsody™ Single-Cell Analysis System from BD Biosciences is a cartridge-based workflow that requires their instrumentation. It isolates cells in individual microwells containing barcoded beads, which then label RNA transcripts upon lysis for library prep. The HT Xpress System can sequence hundreds of thousands of cells per run; drawbacks may include the cost of instrumentation and consumable cartridges.

Parse Biosciences Evercode™ split-pool combinatorial barcoding

With no instrumentation required, Parse Biosciences use a split-pool combinatorial barcoding method to label RNA molecules. Instead of isolating, they begin by fixing and permeabilizing cells, transforming each into their own tiny reaction chamber. “Researchers can profile a wide variety of samples using standard lab tools, making it easy to adopt in any setting, and expand into automation when needed,” says Charlie Roco, Co-founder and Chief Technology Officer at Parse Biosciences. “It also makes the method especially powerful in areas such as cardiology and immunology, where cell populations often vary widely in size and composition.”

Using fixed cells also facilitates sample storage and scheduled processing. “This opens the door to multi-site collaborations, longitudinal studies, and rare sample preservation, while reducing the logistical stress of same-day processing,” says Roco. In addition, the workflow is identical whether running a few samples or millions of cells, so scaling up from pilot experiments is straightforward—the highest capacity Evercode™ Penta kits can sequence up to 5 million cells in one run.

Long-read sequencing

Sometimes length matters in RNA studies. Unlike the methods discussed above, which typically sequence the 3’- or 5’ ends of transcripts in varying lengths, long-read methods can sequence full-length RNA transcripts. Short-read methods are cheaper, widely available, and used in high-throughput studies characterizing cells or tissues, such as immune cell heterogeneity. Sequencing long reads is generally more expensive and lower throughput, but invaluable in certain applications.

Long-read sequencing is currently offered by Oxford Nanopore and Pacific Biosciences (PacBio). Oxford Nanopore detects DNA sequences by measuring changes in an electric current as DNA moves through a nanopore, whereas PacBio reads a DNA sequence from the fluorescent signals generated during its assembly with labeled nucleotides. People are slowly becoming acquainted with long-read options, as short reads were the only sequencing tools available in the early days of NGS. “For years, people thought short-read sequencing was sufficient, but now they’re realizing that genes don’t make proteins—transcripts do,” says Elizabeth Tseng, Associate Director of Product Marketing at PacBio, which offers the Kinnex library prep kit for scRNAseq.

In cancer, tracking mutations that are not located at the ends of genes is only possible with long-read RNA sequencing, says Tseng. “We have customers using single-cell RNA sequencing in cancer to trace clonal evolution or drug-induced mutations, where being able to see the full-length molecule is paramount,” she says. One drawback to long-read sequencing is cost—although the cost has come down recently, it is still higher than short-read sequencing. Though any scRNAseq tool that answers the biological questions you are asking is likely worthwhile.