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A duo for single-cell sequencing
CAMBRIDGE, U.K. & SAN DIEGO—TTP Labtech Ltd, a global designer and developer of automated instrumentation and consumables for life-sciences applications, along with the University of California, San Diego (UCSD), have announced that a collaboration between them has resulted in a new workflow for single-cell sequencing that reduces the cost of sample preparation by as much as 90 percent. The workflow was developed through the combination of UCSD’s expertise in genomics and the use of TTP Labtech’s mosquito liquid handlers, which can pipette nanoliter to microliter volumes with high accuracy and reproducibility, to miniaturize and automate the process.
The article was published in the Journal of Laboratory Automation, and is entitled “Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.”
Dr. Soheila Vaezeslami, senior field application scientists at TTP Labtech, tells DDNews that this collaboration began in early January 2015 “right after the install of the instrument,” adding that “The article became the number one most-read article of the month for the September 2016 issue of JALA.”
Joby Jenkins, product manager at TTP Labtech, commented: “Single-cell sequencing requires large numbers of samples to be handled in order to provide valid data based on population studies. Reproducibility and sensitivity, as well as scalability and cost, can be limiting factors when large numbers of cells are analyzed, and we are very pleased that our partnership with UCSD has achieved our goal of improving sample prep for single-cell genomics research.”
Although single-cell genomics is advancing rapidly, researchers face challenges linked with reproducibility, sensitivity, scalability and cost, particularly when trying to miniaturize high-throughput applications. To maximize accuracy and precision, library prep protocols recommend volumes that are within the range of manual pipettes or large-volume liquid handlers. However, only a small proportion of each of the libraries prepared will be required for sequencing.
The focus of UCSD’s partnership with TTP Labtech was to miniaturize single-cell RNA library prep reaction set-up, to minimize sample and reagent requirements. The resulting workflow has been shown to reduce library prep costs by up to 90 percent while maintaining throughput, accuracy and reproducibility.
“Historically, bottlenecks to this miniaturization process include the inability of standard liquid handlers to accurately dispense volumes under 2 µL and the relatively large volumes needed for the physical nucleic acid shearing steps used in many RNA-seq protocols,” the article states. “We have overcome these barriers by using the mosquito HTS liquid handler, which accurately dispenses volumes between 25 nL and 1.2 µL using true-positive displacement technology in conjunction with an enzyme-based fragmentation method, which can be performed in extremely low-reaction volumes.”
The miniaturization of library prep volumes was achieved using TTP Labtech’s mosquito HTS and HV liquid handlers, which accurately dispense volumes between 25 nL and 1.2 µL and 0.5 to 5 µL, respectively. Both instruments use true positive-displacement pipetting technology, which maintains volumetric accuracy even for highly viscous liquids, enabling high concentration gDNA to be handled, to save on reagent cost and sample input. To assess the quality of the libraries, UCSD applied the workflow to analyze pancreatic differentiation of human embryonic stem cells. The resulting single-cell RNA-sequencing data were analyzed to determine the reproducibility of this system and its ability to distinguish not only between cells at different stages of differentiation but also between individual cells within each stage.
“We used the mosquito HTS liquid handler to complete the [next-generation sequencing] library preparation process using the Nextera XT Library Preparation Kit [from Illumina]. The mosquito HTS uses positive displacement to accurately dispense 25-nL to 1.2-µL volumes, enabling us to scale down the Nextera XT reaction volumes to 2 µL, using as little as 20 pg of input cDNA (compared with a reaction volume of 10 µL and cDNA input amount of 125–375 pg recommended by Fluidigm),” the article says. “This translates to a reduction in input cDNA of fivefold and a cost savings of over fourfold compared with the protocol recommended by the manufacturer, and results in a library preparation cost of less than $1.50 per single cell.”
Dr Louise Laurent, an associate professor at UCSD, said: “The workflow we have developed, in partnership with TTP Labtech, has the potential to provide researchers with significant cost savings while maintaining the quality of single-cell sequencing. The instrument cost and their ease-of-use make the mosquito liquid handlers viable options for labs that would like to scale-down and automate next-generation sequencing sample preparation.”
This isn't the first time UCSD and TTP Labtech have worked together, says Vaezeslami, citing a collaboration “in the crystallography research area, to highlight the importance of mosquito and dragonfly liquid handlers in crystallization of macromolecules in structure-based drug discovery of neglected tropical diseases in the laboratory of Dr. Larissa Podust.”
“Since the mosquito liquid handler is an open platform automatic low-volume multichannel pipettor and independent of chemistry of the kit, and the pipetting technology is based on true-positive displacement, there is no limitation on type of kits, samples or reagents being used on the system, as long as the miniaturization of reaction and sample volumes is possible from a scientific aspect,” Vaezeslami notes. “As long as the reaction volumes can be miniaturized from a data-quality perspective and the application can be run using a multichannel pipettor, mosquito can be used for the same application.”