Large-scale vs. single-cell immunosequencing

Image of cells and DNA to represent comparison of single-cell immunosequencing with large-scale immunosequencing
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    Sequencing an individual’s immune repertoire can enhance our understanding of how the immune system functions and how it responds to disease and treatment. There are a number of methods to profile immune repertoires, most of which focus on sequencing the V(D)J region of T-cell receptor (TCR) and B-cell receptor (BCR) genes. These methods can be broadly categorized as either large-scale immunosequencing or single-cell immunosequencing.

    Both of these techniques provide detailed insight into the immune repertoire. Large-scale immunosequencing via the immunoSEQ® Assay can capture the breadth and depth of a person’s or population’s entire T-cell repertoire, as well as information such as T-cell fraction, clonality, longitudinal dynamics, and repertoire diversity (Figure 1).

    Figure 1. Overview of insights available via large-scale immunosequencing with the immunoSEQ Assay. *COVID mapping is a unique capability of the immunoSEQ Assay via immunoSEQ® T-MAP™️ COVID. Identifying public and private clones is a unique feature of the immunoSEQ Analyzer.

    Both large-scale and single-cell immunosequencing allow you to compare immune repertoires between samples, and identify and track the expansion and contraction of T-cell clones over time. Single-cell immunosequencing, however, can give you information on subpopulations based on transcriptional activity, determine the specific chain pairing for a TCR or BCR, and identify antigen-specific T cells (Figure 2).

    Figure 2. Overview of insights available via single-cell sequencing of immune cells.

    These two approaches differ in terms of sample type and preparation, cost, equipment, data processing, and data output. Choosing the right technique for your research depends on several factors, including your experimental aims and available resources. To help you make an informed choice, we have provided a comparison of these two techniques.

    Single-cell immunosequencing vs. large-scale immunosequencing with the immunoSEQ Assay: How they work

    The immunoSEQ Assay amplifies and sequences complementarity-determining region 3 (CDR3), the most variable region of TCRs and BCRs, for the entire T- or B-cell repertoire. In brief, genomic DNA is isolated from T or B cells, and CDR3 sequences are amplified via a two-step multiplex PCR (Figure 3). The immunoSEQ Assay should not be confused with bulk sequencing, in which entire genomes of all cell types in a given sample are sequenced.

    Figure 3. Overview of large-scale immunosequencing with the immunoSEQ Assay and droplet-based single-cell immunosequencing techniques.

    Single-cell immunosequencing encompasses several different technologies, which differ in the way single cells are isolated and processed. In general, live cells are counted and physically separated via a range of techniques, including flow-activated cell sorting, microfluidics, and laser capture microdissection. (1) One common technique involves mixing cells with barcoded beads to form liquid nano-droplets. The cells are then lysed, and RNA is reverse transcribed to cDNA. The sequences of interest are amplified via PCR and sequenced (Figure 3).

    Large-scale immunosequencing with the immunoSEQ Assay vs. single-cell immunosequencing: Practical considerations

    When choosing between single-cell immunosequencing and large-scale immunosequencing with the immunoSEQ Assay, it’s important to understand the practical requirements and limitations of each technology. Here, we take you through these considerations, from sample type and equipment requirements to data output and analysis.

    Sample types and preparation

    One important consideration when deciding which technology is best for you is the sample types that can be processed.

    The immunoSEQ Assay is highly flexible and is compatible with essentially any sample containing T-cell or B-cell DNA, including sorted B or T cells, cultured cells, peripheral blood mononuclear cells (PBMCs), whole blood, bone marrow, fresh or frozen tissues, or previously isolated genomic DNA. It is notably compatible with notoriously tricky samples, such as formalin-fixed paraffin-embedded (FFPE) tissue or even hematoxylin and eosin (H&E) stained slides. Genomic DNA can be isolated using a wide variety of commercially available kits. While the immunoSEQ Assay is optimized for genomic DNA, it can also be used with cDNA (Figure 4).

    Figure 4. Overview of sample types compatible with single-cell immunosequencing and the immunoSEQ Assay.

    Another benefit of the sample flexibility offered by the immunoSEQ Assay is that samples can be collected and stored before use and existing preserved samples can be utilized.

    In contrast, single-cell immunosequencing is more limited in the range of compatible sample types. Because viable single cells in suspension are required, this method is best suited to samples such as sorted T and B cells. While tissues can be dissociated into a single-cell suspension using a variety of mechanical or enzymatic methods, the need for viable cells means fixed tissues and extracted DNA are not suitable sample types (Figure 4).

    Removing contaminants such as free RNA, dead cells, debris, and clumps is key to ensuring good recovery and data with single-cell immunosequencing. There are a variety of methods for sample cleaning; however, methods that produce the cleanest data, such as thorough centrifugation, can also cause the most sample loss. Therefore, choosing the appropriate sample preparation protocol for your needs often requires balancing speed with sample integrity while also minimizing sample loss. Sample preparation times vary based on the type and quality of the sample but can range from 1 to 1.5 hours for straightforward samples.

    For most single-cell immunosequencing technologies, samples cannot be stored long term and must be processed immediately. (2) It’s generally recommended to analyze cell samples within 30 minutes of preparation to maintain cell viability and limit cell clumping. The requirement for viable cells means that single-cell immunosequencing is not suited to most preserved sample types, including FFPE tissues, H&E-stained slides, or previously isolated and stored genomic DNA.

    Sample size

    The immunoSEQ Assay works well across a wide range of sample sizes, with two main resolutions: Survey and Deep. The Survey resolution can handle samples with between 1000 and 100,000 T cells. For experiments requiring a broader range of the repertoire or requiring greater sensitivity to identify rare clones, the immunoSEQ Assay can achieve Deep profiling resolution with 100,000–200,000 T cells.

    If greater depth is required to achieve insight into large, diverse samples, the immunoSEQ Assay can analyze samples with up to 500,000–600,000 T cells. This Ultradeep profiling resolution is recommended for researchers using our immunoSEQ T-MAP COVID offering.

    While the number of cells required for single-cell immunosequencing varies, most methods require 10,000–20,000 cells per run. (2) Since many of these cells are lost during cell partitioning and isolation, the final yield for single-cell immunosequencing is often much lower than the initial input. The number of cells required also depends on the complexity of the sample. In a heterogeneous, diverse sample, such as a population of T cells all expressing different TCRs, having more cells is preferable. (3)

    Repertoire coverage

    The immunoSEQ Assay provides comprehensive coverage of the immune repertoire. A proprietary internal control system corrects for any PCR bias, allowing absolute quantitative clonal abundance data for T cells and B cells. The available depth of profiling (up to ~600,000 TCRs or BCRs per sample) offered by the immunoSEQ Assay means that it is capable of detecting even incredibly rare clones (e.g. clones with a frequency of 0.001%). (4)

    In contrast, single-cell immunosequencing often provides incomplete coverage of the immune repertoire. In droplet-based methods, capture efficiency is notoriously inefficient, with only about 50–60% of cells captured. (5) In addition, some transcripts are not able to be amplified or detected for even the most sensitive single-cell immunosequencing protocols, a phenomenon known as dropout events. (3,6) Both low capture efficiency and high dropout rates mean that weakly expressed genes and rare clones can be missed. (6)

    Single-cell immunosequencing is also more limited in the number of cells (and therefore the number of TCRs or BCRs) it can profile in a single sample. With the upper limit at around 10,000 cells per library, the depth of profiling is much lower (~50x) than that of the immunoSEQ Assay, making this method less able to detect rare clones.

    Equipment and facility requirements

    Single-cell techniques differ in the equipment and facilities required. There are commercial reagents and platforms for several of the wet-lab steps for single-cell immunosequencing protocols, but the common technologies need custom equipment for cell partitioning and barcoding. (2,3) In particular, droplet-based methods, in which several thousand cells can be assessed, require a dedicated hardware platform.

    In contrast, the immunoSEQ Assay requires no special equipment for library preparation. We offer two options to perform the immunoSEQ Assay: Service or Kit.

    Our immunoSEQ Service is available for human or mouse TCRB, human TCRG, human TCRA/D, and human or mouse IGH loci. We prepare and sequence your samples in-house and send you access to the data via our immunoSEQ Analyzer. All stages of sample preparation, processing, and sequencing are performed by us.

    The immunoSEQ hsTCRB Assay, which sequences the human TCRB locus, is also available as a kit that allows you to prepare your sequencing libraries with less than 4 hours of hands-on time and takes approximately 8 hours from sample to sequencing. The only equipment requirements are a thermocycler to perform PCR.

    Data output and processing

    The immunoSEQ Assay can give insights into several aspects of the immune system, including T-cell fraction, clonality, and repertoire diversity. It also allows you to track clones longitudinally and analyze repertoire overlap between samples. Whether you choose to use the immunoSEQ Service or Kit, your data can be accessed and analyzed via our immunoSEQ Analyzer. This custom-built, user-friendly, web-based data analysis suite allows you to perform a variety of analyses on and between your samples. The intuitive interface allows you to analyze and create publication-ready figures quickly and efficiently.

    Our technical support team is available to assist with all steps of using the Kit, as well as to guide you through data analysis with the Analyzer. For those with more in-depth experience with data analysis, data can be exported for use in alternative analysis software, such as R.

    The immunoSEQ Analyzer allows you to share data and work with colleagues and off-site collaborators in real-time. It is also home to the immuneACCESS® database, where you can access publicly available data and incorporate it directly into your work.

    Single-cell immunosequencing technologies produce vast amounts of sequencing data, which provide the potential for great insight and understanding of the sequenced populations. However, because the data include information from thousands of genes and a large number of cells, single-cell data are noisier, more variable, and more complex than large-scale immunosequencing data. This noise makes it extremely important to choose the right analytical methods to identify and remove low-quality data.

    However, many of the tools designed for bulk sequencing applications cannot be applied to single-cell methods. There are limited computational pipelines for the handling of raw single-cell immunosequencing data and no gold-standard computational software. Data analysis often requires dedicated bioinformaticians or the creation of custom pipelines, which can be costly and time-consuming. (3,6)

    Table 1 below summarizes some of the key attributes of the immunoSEQ Assay and single-cell immunosequencing.

    Table 1: Comparison of single-cell immunosequencing and the immunoSEQ Assay

    Single-cell
    immunosequencing
    Large-scale immunosequencing
    (immunoSEQ Assay)
    Sample requirementsLive cells (>90% viable)Any sample containing T- or B-cell DNA, for example:

    ● Sorted cells
    ● Whole blood
    ● FFPE tissues
    ● H&E-stained slides
    ● Archived samples
    Sample prep● Clean sample: remove debris, dead cells, free RNA, etc.
    ● Determine viability
    ● Measure cell concentration

    ~1–1.5 hours, depending on sample type and quality
    Cell suspensions should be analyzed within 30 minutes of preparation
    ● DNA extraction

    ~20–30 minutes
    DNA can be stored and analyzed at a later point
    Sample inputscDNA prepared after cell partitioninggDNA or cDNA
    Number of cells/receptors sequenced per sampleUp to 10,000Up to 600,000
    Hands-on time for preparing sequencing libraries~3–5 hours~4 hours for Kit
    < 30 mins for Service
    Data analysisMinimal data processing supportimmunoSEQ Analyzer enables a wide array of visualizations
    Expertise and skill required● Understand cell handling requirements to maintain viability and limit clumping
    ● Dedicated hardware platform
    ● Access to DNA sequencing facility
    ● Understand bioinformatics protocols to analyze data
    immunoSEQ Service
    ● None

    immunoSEQ Kit
    ● DNA extraction capabilities
    ● PCR thermocycler
    ● Access to DNA sequencing facility
    Insights● Quantitative transcriptomics
    ● Limited diversity measurements
    ● Cannot track rare clones
    ● Quantitative, comprehensive TCR repertoire information
    ● Accurate diversity metrics
    ● Longitudinal clone tracking

    Choosing the right immunosequencing technology to answer your research question

    Through comparing immunoSEQ Technology with single-cell immunosequencing it is clear that both technologies have their advantages and limitations. The technology you choose depends on your available resources and research questions. It is possible to use both technologies to gain deeper and broader insights. However, in some instances, large-scale immunosequencing may be a better choice, especially for identifying rare clones or tracking the immune repertoire over time. Below are two examples that show the insights you can uncover with immunoSEQ Data.

    Tracking the circulating follicular helper T-cell response after annual influenza vaccinations

    In this study, the immunoSEQ Assay was used track a subset of circulating follicular helper T cells (cTfh) induced by the influenza vaccine and associated with increased plasmablasts. (7) 

    T follicular helper cells (Tfh) located in the germinal center, play a key role in B-cell activity and responses to antigens and antibody production. The authors identified a subset of cTfh that express high levels of inducible costimulatory (ICOS) and CD38. These ICOS+CD38+ cTfh cells were identified as the activated pool because they expressed many proteins associated with T cell activation and other proteins that are necessary for cTfh activity. The population of ICOS+CD38+ cTfh expanded after influenza vaccination and this population showed influenza-specific responses seven days after vaccination.

    The immunoSEQ Assay was used to examine the clonality of ICOS+CD38+ cTfh, which increased in most participants after influenza vaccination. Longitudinal analysis showed a high turnover in the activated cTfh pool at baseline in the absence of vaccination. However, repeated flu vaccination led to re-emergence of influenza-specific memory clonotypes at day 7 after vaccination in years 2 and 3 in the activated cTfh (Figure 5A). The stable ICOS-CD38- cTfh subset may represent a memory pool from which antigen-specific Tfh cells could be recalled following antigen exposure (Figure 5B).

    Figure 5. (A) Clonality changes in the subsets over time. The increase in clonality of the activated ICOS+CD38+ cTfh cells suggests an antigen-driven clonal expansion following vaccination, while the potential memory subset remains consistent, even following vaccination. (B) Overlap of ICOS+CD38+ (activated) cTfh and ICOSCD38 cTfh in patient 999. Successive vaccination-induced similar repertoire responses each year in the ICOS+CD38+ subset, while the ICOSCD38 subset remained fairly stable, likely acting as a memory pool. Adapted from Herati et al. (7)

    Insight into the clonal kinetics of chimeric antigen receptor T-cell therapy using the immunoSEQ Assay and single-cell immunosequencing

    In this study, the power of the immunoSEQ Assay was used to probe the clonal kinetics of chimeric antigen receptor (CAR) T-cell therapy in cancer patients, in combination with single-cell immunosequencing methods. (8) The immunoSEQ Assay was used to see how the CAR T-cell population changed over time. CAR T cells were sequenced before infusion and at 1 week and 1 month after infusion. Results from this large-scale TCRβ sequencing showed that the initial clonal composition of the infused cells does not predict the clonotypes that will persist in the host; some initially rare clones expanded and came to dominate the population. Single-cell immunosequencing was used to transcriptionally characterize the clones that eventually expanded in the patient. The clones that expanded the most post infusion expressed high levels of genes involved in cytotoxicity and proliferation (Figure 6).

    This study highlights a key advantage of the immunoSEQ Assay over single-cell sequencing: the ability to track T-cell clones over time and across different tissues. The fast turnaround time and user-friendly data-analysis suite, the immunoSEQ Analyzer, make it easy to analyze a large number of samples. In contrast, conducting single-cell immunosequencing assays at different time points and for different samples is often cost- and labor-intensive.

    Figure 6. Results from Sheih et al. (8) showing (A) how many of the top 10 clones in the early time point originated from low-ranking clones in the infusion assay (determined using the immunoSEQ Assay) and (B) the fraction of transcriptionally distinct clusters among CD8+ CAR T cells in the infusion product (IP) that were either detected or not detected at three time points following infusion (determined using single-cell immunosequencing). Adapted from Sheih et al. (8)

    Large-scale immunosequencing with the immunoSEQ Assay vs. single-cell immunosequencing summarized

    Table 2 summarizes the key differences between the immunoSEQ Assay and single-cell immunosequencing.

    Table 2: Single-cell vs. large-scale immunosequencing

    Single-cell immunosequencingThe immunoSEQ Assay
    Range of suitable sample inputs++++
    Cost$$$$
    Hands-on time++++
    No specialized equipment required++++
    Ability to detect rare clones++++
    Scalability++++
    Ease of data analysis++++

    This table highlights that while both the immunoSEQ Assay and single-cell immunosequencing are powerful techniques, there are key differences in the ability to process different sample types, the high cost and the need for specialist equipment for single-cell immunosequencing, and the power to detect rare clones. Therefore, when designing your immunosequencing experiment, you need to carefully consider the practical and technical differences between these two techniques to achieve your experimental goals within your budget.

    Our scientists can help you decide if the immunoSEQ Assay is right for your needs and, if so, provide end-to-end support from designing your experiments to analyzing your data.

    Contact us to learn how the immunoSEQ Assay can help propel your research.

    For Research Use Only. Not for use in diagnostic procedures.

    References

    1.    Hwang B, Lee JH, Bang D. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp Mol Med 2018;50:96.

    2.    See P, Lum J, Chen J, Ginhoux F. A Single-Cell Sequencing Guide for Immunologists. Front Immunol 2018;9:2425.

    3.    Haque A, Engel J, Teichmann SA, Lönnberg T. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications. Genome Med 2017;9:75.

    4.    Robins H, Desmarais C, Matthis J, Livingston R, Andriesen J, Reijonen H, et al. Ultra-sensitive detection of rare T cell clones. J Immunol Methods. 2012;375(1–2):14–19. doi:10.1016/j.jim.2011.09.001

    5.    Chiffelle J, Genolet R, Perez MAS, Coukos G, Zoete V, Harari A. T-cell repertoire analysis and metrics of diversity and clonality. Curr Opin Biotechnol 2020;65:284–295.

    6.    Chen G, Baitang N, Tieliu S. Single-Cell RNA-Seq Technologies and Related Computational Data Analysis. Front Genet 2019;10:317.

    7.    Herati RS, Muselman A, Vella L, Bengsch B, Parkhouse K, Del Alcazar D, et al. Successive annual influenza vaccination induces a recurrent oligoclonotypic memory response in circulating T follicular helper cells. Sci Immunol. 2017;2(8):eaag2152.

    8.    Sheih A, Voillet V, Hanafi LA, DeBerg HA, Yajima M, Hawkins R, et al. Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy. Nat Commun 2020;11:219.

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