Single-Cell Transcriptome Analysis on FFPE Samples: A Comparative Study with M20 VITA 

Pathological research assays, including histological, IHC, and in situ genomics analyses, frequently utilize formalin-fixed paraffin-embedded (FFPE) samples. Biorepositories and pathology departments worldwide house millions of these samples, offering an extensive collection of readily available specimens for in-depth analysis. However, despite their prevalence, FFPE samples pose challenges due to molecular cross-linking and nucleic acid degradation during preservation.

Single-cell RNA Sequencing (scRNA-seq) on clinical FFPE specimens promises breakthroughs in precision diagnostics, treatment strategies, and prognostic insights for human diseases. However, challenges persist in isolating intact cells or nuclei and capturing RNA from FFPE tissues.

Until today, few commercial platforms are available that enable scRNA-seq in these precious but challenging samples. With the introduction of M20 VITA technology in August 2022, alongside our VITA product series, we've provided a solution to access the valuable information stored in these samples. Using random primers, M20 VITA overcomes the boundaries of  other scRNA-seq technologies that mostly rely on poly(A) tails for RNA capture. As these structures are prone to chemical modification and degradation, the capabilities to obtain quality data from FFPE samples using technologies based on poly(A) capture are limited.

In parallel, 10x Genomics introduced the Chromium Single Cell Gene Expression Flex  (from here on called 10x Flex), a probe-based assay for whole transcriptome expression, enabling single-cell transcriptome sequencing of FFPE samples.

While both platforms provide solutions for single-cell transcriptome analysis in FFPE samples, M20 VITA distinguishes itself with its unique approach to capturing full-length transcripts, ensuring researchers obtain comprehensive and reliable data of high quality.

M20 VITA and 10x Flex Technologies: An Overview

The RNA capture presents a critical step in scRNA-seq technologies, significantly influencing the breadth and depth of insights derived from transcriptome profiles. Understanding the technical nuances of these methods is crucial to choose the optimal technology for the project's aims.

The M20 VITA workflow begins with fixation, permeabilization, and the blocking of single-stranded DNA, ensuring the preservation of the cell's biological state and preparing it for in situ reverse transcription. The reverse transcription step employs random primers (Figure 1), enabling the comprehensive capture of the cell's entire transcriptome. Additionally, an adapter is added to the cDNA strand, facilitating the subsequent barcoding of individual cells. Following this step, amplification, library generation, and sequencing are conducted, and the obtained data are ready for analysis.

Figure 1: Illustration of the M20 VITA Principle

In contrast, the 10x Flex assay relies on probe hybridization for RNA capture, targeting around 18,000 genes. The significant difference in RNA capture methodologies between the two technologies leads to diverse features and capabilities (Table 1). M20 VITA's unique random primer approach covers a wide range of species without restrictions. By randomly hybridizing to transcripts, it isn't confined to targeted mRNA sites, allowing for the capture of over 30,000 genes in human samples. Moreover, it facilitates the analysis of a broad spectrum of RNA biotypes.

Table 1: Comparison of M20 VITA and 10x Flex Technologies

Selecting the most suitable technology is a critical aspect of planning any experiment. M20 VITA stands out for its broad compatibility with various sample types and species, along with its comprehensive transcriptome capture capabilities. Beyond meeting basic requirements such as sample type or species compatibility, the choice of the optimal platform depends on its efficacy. To assess this thoroughly, we engaged a third-party institution to employ both M20 VITA and 10x Flex technologies for scRNA-seq on a FFPE prostate cancer tissue sample preserved for two years.

Performance and Benefits: Evaluation of M20 VITA Against the Alternative Technology

1. Performance and Data Quality

When comparing the metrics of both datasets (similar data volumes), 10x Flex libraries achieve a median gene count of 1,243 and a total gene detection of 15,558 at a sequencing saturation of 92.79%. In contrast, M20 VITA displays a saturation of 57.45%, along with a median gene count of 1,002 and a total gene detection of 31,141. Increasing the sequencing depth of theM20 VITA library leads to a median gene count of 1,287, resulting in a modest rise in sequencing saturation to 65.5% (Table 2). These results highlight thatM20 VITA provides data quality comparable to 10x Flex at significantly lower sequencing saturation.

Table 2: Sequencing metrics of M20 VITA and 10x Flex Libraries

The gene saturation curve shows that 10x Flex has reached a plateau, while M20 VITA displays significant room for growth (Figure 2), suggesting that M20 VITA holds promise to further enhance detection capabilities with increasing sequencing depth.

Figure 2: Saturation Curve for M20 VITA and 10x Flex Libraries

2. Sensitivity of Gene Detection

M20 VITA successfully captured a total of 31,141 genes, whereas 10x Flex detected 15,558 genes (both datasets exclude mitochondrial and ribosomal genes, and those expressed in fewer than three cells). The overlapping genes detected by both technologies numbered 14,357. M20 VITA remarkably identified 16,784 unique genes, whereas 10x Flex detected only 1,201 (Figure 3). This striking disparity underscores M20 VITA's excellent performance, detecting 15,583 more unique genes than 10x Flex.

Figure 3: Number of Captured Genes with M20 VITA and 10x Flex

3. Full-length Coverage

The innovative random primer-based capture technology of M20 VITA ensures unbiased coverage across the entire gene body. Nevertheless, in FFPE samples where degradation primarily impacts the 3' end, there is a discernible inclination towards increased coverage at the 5' end. Conversely, 10x Flex exhibits a notable reduction of coverage, particularly evident at the 3' end (Figure 4).

Figure 4: Comparison of mRNA 5'-3' Gene Body Coverage between M20 VITA and 10x Flex

At the level of individual cell nuclei, M20 VITA demonstrates enhanced genome coverage in comparison to 10x Flex (Figure 5).

Figure 5: Comparison of Genome Coverage between M20 VITA and 10x Flex

At the single-gene level, the read distribution highlights another contrast between the probe-based technology and a random primer-based approach. M20 VITA reads span across the majority of genes, whereas 10x Flex reads are limited to probe-targeted regions of less than 100 base pairs (Figure 6).

Figure 6: Comparison of Gene Coverage between M20 VITA and 10x Flex

4. Cell Type Annotation Based on mRNA

Leveraging the expression data of the 3000 most highly expressed mRNAs for cell clustering, M20 VITA delineated 18 distinct cell populations (refer to Figure 7). Comparative analysis between the two platforms underscores the capability of M20 VITA to detect an additional cell population, indicative of its enhanced sensitivity. These findings underscore the superior resolution of M20 VITA in unraveling cellular heterogeneity.

Figure 7: Identification of Cell Populations on Data Obtained with M20 VITA and 10x Flex

While M20 VITA achieves higher resolution in detecting cell populations, it maintains consistency in annotation, similar to the 10x Flex platform (Figure 8).

Figure 8:  mRNA-based Cell Type Annotation on Data obtained with M20 VITA and 10x Flex

5. Detection of Non-Coding RNA

5.1  Proportion Analysis of lncRNA

The unique RNA capture approach of M20 VITA facilitates the detection not only of mRNA but also various RNA biotypes, including long non-coding RNAs. Analysis of datasets obtained with M20 VITA unveiled a detection range of 6% to 30% of lncRNA within each cell. In contrast, the probe-based 10x Flex Assay exclusively enables targeted mRNA transcript detection (Figure 9).

Figure 9: Proportion of Detected lncRNA with M20 VITA and 10x Flex

5.2 Cell Type Annotation Based on lncRNA 

Utilizing SingleR ^[1] for cell type annotation based solely on the 3000 most highly expressed lncRNAs showed that integrating lncRNA data from M20 VITA improved annotation accuracy. This integration unveiled additional cell types like neutrophils and smooth muscle cells (Figure 10), which were not identified with 10x Flex (compare with Figure 8).

Figure 10:  Cell Type Annotation based on lncRNA with M20 VITA

5.3 Cell Type Annotation Based on mRNA and lncRNA 

The clustering based on the expression data of the 3000 most highly expressed transcripts of both mRNA and lncRNA in M20 VITA data discerned 22 distinct cell populations (Figure 11, left). This count surpasses that achieved with mRNA-based clustering by 4 cell populations and exceeds 10x Flex by 5 (compare Figure 8).

Notably, the identified cell types aligned with the ones annotated based solely on lncRNA. These findings not only affirm the reliability of lncRNA-based annotation but also underscore the promising potential of M20 VITA in elucidating the substantive significance and specificity of lncRNAs.

Figure 11: Identification of Cell Populations (left) and Cell Type Annotation (right) Based on mRNA and lncRNA of Data obtained with M20 VITA

 Setting a New Benchmark: M20 VITA's Performance in Single-Cell RNA Sequencing of FFPE Samples

Choosing the right technology is crucial for achieving optimal results in scientific projects. The evaluation of M20 VITA against the sole commercially available alternative for analyzing FFPE samples with scRNA-seq underscores it‘s versatility and its numerous advantages. Notably, M20 VITA excels in sensitivity, coverage and capture of RNA types and  cell type annotation.

M20 Genomics's cutting-edge products unlock novel and unparalleled insights from FFPE samples, tapping into a wealth of novel information. These capabilities hold significant implications for clinical studies, long-term and retrospective investigations, and further research avenues in pivotal fields such as oncology or immunology. This advancement paves the way for groundbreaking discoveries and advancements, enhancing our understanding of human health and disease.

* For further inquiries or more information about M20's innovative products, please don't hesitate to reach out to us at info@m20genomics.com. We are committed to pioneering innovation and welcome opportunities to collaborate and contribute to  advancing our understanding of human health and disease.

Reference

[1] Aran D, et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nature immunology. 2019;20(2):163–172.