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Case Studies in Unlocking Biological Insights Through Automation-Enabled Single-Cell Sequencing

By SPT Labtech

Single-cell sequencing offers unprecedented resolution into cellular heterogeneity and has enabled researchers to unravel complex biological systems and gain critical insights that were previously invisible to traditional bulk sequencing approaches. By probing the genome and transcriptome within individual cells, researchers can identify rare and unique cell types, elucidate developmental lineages, and uncover tumor composition. As the technology evolves, single-cell analysis promises to transform personalized medicine through early diagnostic testing and gene therapy targeting.

The limitations of manual single-cell workflows

Despite its potential, single-cell analysis poses significant throughput challenges in generating the vast datasets necessary for comprehensive biological study. Unlike bulk sequencing, which analyzes entire tissues in a single reaction, single-cell techniques traditionally require individual processing of each cell. This drastically increases the reagents and sequencing reads needed to gain adequate coverage of the genome or transcriptome at a cellular resolution. Moreover, the complexity of biological systems demands the analysis of hundreds to over a million cells to fully characterize tissue composition and delve into subpopulation biology.

Manual RNA sample and library preparation for DNA or RNA sequencing at this scale is incredibly tedious and expensive, limiting the accessibility of single-cell analysis for many labs. While there are a variety of novel technologies to facilitate high-throughput single-cell experiments, plate-based methods remain a vital tool to answer a wide range of biological questions and are often complimentary to other technologies. This discussion focuses on examples of such plate-based methods and how cost-effective and robust workflows can be realized with automated miniaturized technology.

The role of automated positive displacement liquid handling

Automated liquid handling solutions drive speed and efficiency in library generation, reducing the time required for high-quality library generation to as little as two hours. By removing the burden of manual pipetting, scientists can instead dedicate time to tasks where their experience and insight are of more value. Automation enables running protocols in 96- or 384-well formats simultaneously, significantly improving throughput in library generation. This accelerates data generation and scalability without imposing significant resource costs. 

Flowchart showing the workflow for single cell RNA-seq, single cell DNA-seq, bulk RNA-seq, and bulk DNA-seq, followed by library preparation. The steps are as follows:  Single cell RNA-seq:  Prepare lysis plate Isolate single cells Generate cDNA PCR Bead clean-up Quantification and normalization Library preparation Single cell DNA-seq:  Prepare lysis plate Isolate single cells PCR Bead clean-up Quantification and normalization Library preparation Bulk RNA-seq:  Extract RNA (high volumes) Quantification and normalization Library preparation Bulk DNA-seq:  Extract gDNA (high volumes) Quantification and normalization Library preparation The library preparation step includes:  Fragmentation, end prep, A-tailing Ligation PCR Bead clean-up Quantification and normalization Pooling
Figure 1. Standard sample and library preparation workflow steps for single and bulk DNA and RNA sequencing. All steps in teal and pink can be automated and miniaturized effectively using positive displacement liquid handling.

Positive displacement technology has emerged as a hero technique. Unlike air displacement technology, found in handheld pipettes and even in standard automated systems, platforms utilizing positive displacement technology are not disturbed by air pressure as a piston comes into direct contact with the liquid without an air cushion. This maintains accuracy and precision across all liquids from viscous to volatile, down to nanoliter volumes.

Substantial cost savings through reaction miniaturization

To assure high accuracy and precision, most library preparation protocols recommend volumes that are within the range of manual pipettes or of large-volume liquid handlers. However, only a fraction of each library volume is required for sequencing.

Therefore, one way to save costs on expensive reagents is through reaction miniaturization. While it may only be feasible to reduce volumes down to half of the recommended volumes when pipetting by hand, automated positive displacement systems enable more aggressive miniaturization, down to 50 times smaller than the recommended volumes depending on the assay chemistry. The ability to generate high-quality libraries at a fraction of the volume makes single-cell techniques a financially viable option for more laboratories, lowering the barrier to large-scale and high-throughput studies.

Additionally, in single-cell studies, the heightened resolution from smaller volumes translates to the detection of more genes and finer genetic details, enriching the quality of data.

Value in versatility

In such a dynamic field, new methods and library preparation kits are constantly emerging. As such, the most valuable automated systems are those that can easily accommodate new chemistries. Again, positive displacement technology proves advantageous here with consistently high accuracy across even the most viscous and challenging liquids. This versatility ensures compatibility across various kits and reagents, maintaining flexibility in experimental approaches. Its value has already been demonstrated in a range of applications across genomics and drug discovery.

Case study #1: Development of novel allergy therapeutics

IgGenix, a preclinical discovery and development company creating novel therapeutics against allergies with re-engineered antibodies, uses single-cell transcriptomics to isolate rare IgE-producing B cells from individuals with severe allergies. Single-cell RNA sequencing recovers full-length paired heavy and light chain sequences that compose monoclonal IgE antibodies, discovering disease-causing antibodies in allergic individuals. These are then re-engineered as therapeutics that retain their ability to bind to the allergen.

Single cells are required for this research as bulk methods lose critical pairing information needed to reconstruct antibodies. Positive displacement liquid handling — using a combination of mosquito multichannel pipetting, mixing, and pooling, and dragonfly non-contact dispensing — has been successfully used to achieve the necessary throughput in sample and library preparation for plate-based next-generation sequencing, as well as miniaturizing assays by up to 20-fold to deliver significant reagent cost savings.

Case study #2: Generation of cell atlases to uncover gene expression insights

Researchers from Chan Zuckerberg Biohub San Francisco have automated single-cell RNA sequencing to generate transcriptomics maps, or atlases, for different model cell types, including human and zebrafish. These atlases provide valuable insights into gene expression patterns within specific cells and how this expression dictates specialized cellular function in healthy and diseased tissues and organs.

Due to the sheer scale of library preparation work required in building an atlas of this kind, automation has allowed researchers to make more efficient use of time and reagents, streamlining otherwise tedious tasks:

— Column dispensing and pooling on mosquito.

— Reagent dispensing during cDNA synthesis using dragonfly.

— Bead clean-up, library preparation, pooling, stamping, and normalization on firefly.

This sponsored content is provided by an advertiser and published in collaboration with the GW Custom Solutions Group, a division of GenomeWeb. The content was not produced by the editors or reporters of GenomeWeb, 360Dx, or Precision Oncology News, and does not represent the views of these publications or GenomeWeb's parent company, Crain Communications Inc.