Broadening doors to use non-ideal samples to achieve meaningful and rich single-cell multiomics data through an advanced sequencing technique and expert collaborations

Author: Joseph Modarelli

In today’s competitive and fast-paced drug development landscape, securing meaningful and rich insights from samples with compromised viability or quality is critical in driving important clinical trials forward for patients in need. Traditionally, extracting clinically relevant data from non-ideal samples was limited and often avoided. Given the complexities of today’s clinical research and the limited accessibility to disease- or population-relevant sample types, clinical trial investigators may not have sufficient control over sample quality, leading to clinical samples with impacted cell viability or availability.

The good news is that rich single-cell multiomics data from quality-impacted samples has become achievable through recently developed advanced techniques. For example, Cellular Indexing of Epitopes and Transcriptomes by Sequencing (CITE-Seq) is a technique that has been widely adopted for biomarker discovery ahead of clinical trials to simultaneously interrogate transcriptional and surface protein data from a single cell. However, when integrating emerging single-cell multiomic techniques like CITE-seq, it is still important to incorporate processes to maximize sample quality (e.g., age, viability, cell recovery, etc.) to obtain the best possible data from a patient sample. This includes providing clinical trial teams and expert lab providers with continuous technical guidance and vendor support on the use of CITE-seq and understanding practical limitations in analyzing data from multiple lab sites to optimize parameters.

Examining CITE-Seq reproducibility

Through a strategic partnership, BioLegend, a global developer of cutting-edge antibodies and reagents, collaborated with IQVIA Laboratories, as the first and only certified service provider to offer this capability, to demonstrate the reproducibility of its CITE-Seq single-cell product on the 10x Genomics Chromium platform, while also providing guidance for clinical sample collection ahead of downstream processing.

For the evaluation, the expert teams used matched donor cryopreserved human peripheral blood mononuclear cells (cPBMCs) and bone marrow mononuclear cells (cBMMCs), two common human sample types that offer high clinical relevance for human disease and represent routine sample types used for clinical evaluations. cBMMCs can be technically challenging to work with and the quality of both sample types can be highly variable depending on source, specimen, and handling (storage, time, operator, etc.).

Steps taken across sites

In evaluating sample reproducibility, matched cPBMCs and cBMMCs samples from three donors were acquired and run in parallel at both BioLegend and IQVIA Laboratories with some key steps matched at both sites:

• All six donor samples were stained using BioLegend TotalSeq™-C Universal Antibody Cocktails (PN 399905) and additional clinically relevant TotalSeq™-C antibody markers CD235ab, CD90, CD34, CD10, CD135, CD117, CD206, CD271, CD70, CD9, CD200 and CD74.
• cBMMCs were processed using identical procedures across both physical locations to investigate inter-site reproducibility.

The only unique step was that IQVIA Laboratories artificially created clinically low-quality cPBMCs by “cold shocking” half of the matched samples to simulate reduced sample quality.

Demonstrating consistency with quality

In examining the CITE-Seq technique using the 10x Genomics Chromium platform, results generated at IQVIA Laboratories and BioLegend sites using different equipment and operators showed a very high degree of technical repeatability between each replicate per sample (two replicates per sample as shown in this poster).

In terms of cell viability, the cPBMCs samples that underwent the temperature shocking method demonstrated reduced cell viability and 10x Cell Ranger antibody metrics, as expected. However, CITE-seq data for cPBMCs were still usable and revealed many insights into cell types, protein, and RNA metrics when compared with donor-matched normally processed (i.e. high-quality) samples. Also as expected, cBMMCs had a lower cell viability before loading the 10x Chromium instrument but demonstrated high correlation between study sites and matched donor samples for key downstream quality control metrics.

Evaluation findings emphasize how CITE-Seq using BioLegend’s TotalSeq reagents is a viable solution for clinical samples of potentially varying quality. With optimized and standardized operational processes and related expert oversight, it is feasible to obtain high-quality data from less than ideal clinical samples. See Figure 4 from the poster noted above:

Click here to learn more about IQVIA Laboratories Genomics (formerly known as Q² Solutions) single-cell technologies, capabilities and expertise to offer multiomics solutions for researchers, ensuring non-ideal samples can be useful for quality and meaningful data insights.