Single-cell RNA sequencing (scRNA-seq) has revolutionized our ability to examine the cellular heterogeneity of complex samples, identify new cell types, and explore transcriptional regulation at unprecedented resolution. But not all scRNA-seq methods are created equal, and the right choice depends heavily on your biological question, sample type, and experimental design.
In this article, we provide a practical comparison of MERCURIUS™ FLASH-seq from Alithea Genomics and 10x Genomics Chromium scRNA-seq technologies to help you understand their unique strengths and limitations that shape their suitability for different experimental goals.
MERCURIUS™ FLASH-seq vs 10x Genomics Chromium at a Glance
Both MERCURIUS™ FLASH-seq and 10x Chromium produce high-quality single-cell transcriptomic data, but they differ significantly in their sequencing strategy, throughput, data output, and best-use scenarios.
The choice of method depends on your scientific question: Do you need depth (fewer cells with richer transcript data) or breadth (many cells with relatively shallow profiling)?
- MERCURIUS™ FLASH-seq for depth — a 384-well plate-based, full-length scRNA-seq method optimized for speed, sensitivity, and deep insights at isoform resolution that’s suited to rare cell types, low numbers of isolated cells, or low-input RNA samples.
- 10x Genomics Chromium for breadth — a droplet-based, 3’ or 5’ end-tagging technology for high-throughput profiling of thousands of cells in parallel.
Here’s a quick overview of some core differences before we dive into specifics:
Table 1. Comparison of key technological and protocol differences between MERCURIUS™ FLASH-seq and 10x Chromium scRNA-seq technologies. Genes detected per cell vary by sequencing depth, so head-to-head comparison at the same read depth taken from (1).
Core Workflow and Technology Differences
1. MERCURIUS™ FLASH-seq allows fine control of input cell types
The workflow of plate-based MERCURIUS™ FLASH-seq differs from emulsion droplet methods like 10x Chromium, as it first requires users to deposit single cells into 384-well plates via FACS or other dispensing methods, or to manually include low-input RNA samples that are challenging to sequence with other methods. This makes the technology optimal for small cell populations of less than 1000 cells. In contrast, 10x Chromium methods generally require a minimum of 1000 cells from a high-quality single-cell suspension, either sorted or directly dispensed, and are best suited to samples where you can generate many healthy, dissociated cells, like PBMCs or heterogeneous embryonic tissues.
The use of FACS or other single cell dispensing methods allows the precise isolation of cell types of interest, like CD8+ naïve T cells or other immune cell subtypes, for subsequent deep transcriptomic profiling. This prior isolation is crucial for precious or rare populations when only relatively small numbers of cells are present. Even in heterogeneous hPBMCs with low RNA content, FLASH-seq enables the sensitive detection of different immune cell types (1).
10x Chromium technology relies on bulk droplet capture, where you have limited control over which subpopulations are loaded. Furthermore, at least 20% of cells are lost in droplet-based methods due to inefficient encapsulation, cell type viability, or data filtering issues (2). This loss could reduce library complexity, compromise data quality, and could ultimately be unacceptable for studies lacking sufficient cell numbers in the first place. Individual sorting and processing in 384-well plates guarantees maximum recovery.
2. MERCURIUS™ FLASH-seq Chemistry: Sensitive full-length data
MERCURIUS™ FLASH-seq uses a fundamentally different workflow from the 10x Chromium technology, as it is based on the Switching Mechanism at the 5′ end of RNA Template approach used by the popular Smart-seq2/3 methods (3,4). The original FLASH-seq protocol was published in 2022, but Alithea’s MERCURIUS™ version has improved upon this with a non-toxic tagmentation buffer, dyes for QC of stages like lysis and Tn5 inactivation, and optimizing for ultra-low RNA inputs of 1pg to 1 ng (1). It also offers improved speed and sensitivity by combining the RT and TSO-based pre-amplification reactions into one RT-PCR reaction. These plate-based approaches have been fundamental tools for researchers requiring full-length scRNA-seq that provides robust information about transcript isoforms, alternative promoter usage, and SNP detection not previously possible with droplet-based methods.
Please see our other blog posts for a complete overview of FLASH-seq and a comprehensive comparison of FLASH-seq and MERCURIUS™ FLASH-seq vs Smart-seq2/3.
As a result of extensive method optimization, FLASH-seq dramatically improves upon speed and sensitivity compared to previous Smart-seq methods by combining the RT and TSO-based pre-amplification reactions into one RT-PCR reaction. It is recommended by an independent benchmarking study of ten scRNA-seq technologies for researchers performing many experiments with low numbers of cells (the same study recommends 10x Chromium for experiments that require high cell numbers) (5).
3. 10x Chromium Chemistry: Droplet-based tagging
In contrast to plate-based scRNA-seq methods, the 10x Chromium system uses microfluidics and oil droplets to co-encapsulate individual cells with gel beads coated in barcoded oligonucleotides (2). These oligonucleotides contain cell-specific barcodes, unique molecular identifiers for mRNA molecule counting, and a poly(dT) stretch to capture mRNA 3′ ends (5′ capture is also possible depending on the kit used). Reverse transcription barcodes the end of transcripts originating from the encapsulated cell. This tagging method allows for subsequent quantification of transcript numbers and cell-by-cell gene expression profiling.
10x’s Next GEM protocols capture the 3′ or 5′ end of polyadenylated RNA molecules, resulting in high-throughput profiling of gene expression counts but limiting information on transcript isoforms or complete gene structure. However, the recent launch of GEM-X Chromium chemistry suggests that full-length transcript readouts are now possible (2).
4. Depth versus throughput?
While the throughput is lower than emulsion droplet-based methods like 10x Chromium, MERCURIUS™ FLASH-seq in 384-well plate format provides a much deeper analysis of the transcriptome as standard. In a head-to-head comparison at the same sequencing depth, FLASH-seq detected an average of around 6,000 genes, including low-abundance transcripts and those with higher GC content or longer exonic sequences, versus around 2,500 for 10x’s Next GEM kit (1). If sequencing depth increases, MERCURIUS™ FLASH-seq can detect up to 12,000 genes per cell if required.
Furthermore, as the data generated by MERCURIUS™ FLASH-seq is full-length, it enables the characterization of cell clusters, differential gene expression, or cell type identification, alongside the detection of splice isoforms, allelic variants, and single-nucleotide polymorphisms that are challenging to achieve with higher throughput, but arguably lower resolution methods.
Despite its lower average gene detection rate, 10x Chromium excels at large-scale, broad surveys, as users can profile tens of thousands of cells in a single experiment from heterogeneous samples. The data consists of gene expression matrices, suited for cell clustering, differential gene or cell type identification, and mapping tissue complexity. Previous iterations of the Chromium technology gave only 3’ or 5’ read-outs with limited visibility into alternative splicing, transcript variants, or cis-regulatory variation. The new Universal 3' and 5’ Gene Expression kits based on GEM-X chemistry now aim to overcome these challenges.
5. Additional transcriptomic information aids discovery
Comparative experiments using human retinal organoids exemplify the strength of the original FLASH-seq protocol. While 10x Genomics Next GEM enabled broad reconstruction of cell lineage progression, FLASH-seq enabled more precise cell type annotation due to a higher information content per cell and facilitated confident identification of cell-type marker genes and detection of splice variants. For instance, it distinguished between PKM1 and PKM2 isoforms of pyruvate kinase, which 10x data could not resolve (1). FLASH-seq also allowed direct SNP calling at single-cell resolution, with a greater true-positive detection rate compared to variant calling from aggregated 10x data at similar sequencing depths (1).
FLASH-seq data also allows immunology researchers to infer T-cell receptor (TCR) and B-cell receptor (BCR) sequences, including the critical antigen-binding CDR3 amino acid (CDR3aa) region, even at low sequencing depths (1). This multimodal information enables users to pair immune receptor identity with the full transcriptomic state of the same cell without requiring high sequencing depth or multiple specialized protocols.
Use Cases and Research Applications
Both platforms allow researchers to interrogate cellular complexity, but the nature and utility of their data diverge. By understanding these distinctions and aligning technology choice with specific research questions, scientists can maximize discovery and insight from their single-cell experiments.
The sensitivity of MERCURIUS™ FLASH-seq makes it optimal for studies requiring:
- Deep transcriptomic insight into rare cell types of interest
- Detailed isoform-level analysis and alternative splicing discovery
- Investigating allelic expression and SNPs at single-cell resolution
- High-sensitivity detection of low-abundance transcripts
- Small to medium-scale experiments where depth and diversity are preferable to ultra-high cell throughput
Example use case: If you’re studying how a rare mutation alters isoform usage in different cell types from a small patient biopsy, MERCURIUS™ FLASH-seq is ideal.
10x Chromium’s throughput make it ideal for:
- Building cell atlases across tissues or developmental stages
- Rapid screening for marker genes, cell states, or population structures
- Studies identifying rare cell types in heterogeneous samples
Example use case: If you’re mapping developmental trajectories across 50,000 cells in an embryo, 10x Chromium is suitable.
Interested to learn how MERCURIUS™ FLASH-seq could accelerate your next small- to mid-scale scRNA-seq project? Schedule a free consultation call or contact us to find out more.
References
- Hahaut V, Pavlinic D, Carbone W, Schuierer S, Balmer P, Quinodoz M, Renner M, Roma G, Cowan CS, Picelli S. Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq. Nature biotechnology. 2022 Oct;40(10):1447-51.
- 10x Genomics. Universal 3′ Gene Expression. Available at: https://www.10xgenomics.com/products/universal-three-prime-gene-expression.
- Picelli S, Faridani OR, Björklund ÅK, Winberg G, Sagasser S, Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. Nature protocols. 2014 Jan;9(1):171-81.
- Hagemann-Jensen M, Ziegenhain C, Chen P, Ramsköld D, Hendriks GJ, Larsson AJ, Faridani OR, Sandberg R. Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nature biotechnology. 2020 Jun;38(6):708-14.
- Hornung BV, Azmani Z, den Dekker AT, Oole E, Ozgur Z, Brouwer RW, van den Hout MC, van IJcken WF. Comparison of single cell transcriptome sequencing methods: of mice and men. Genes. 2023 Dec 16;14(12):2226.
