New next-generation single-cell approach offers absolute quantification of gene expression with orders of magnitude improvement in throughput and cost
A publication in the journal Science demonstrates a new, massively parallel technology to interrogate gene expression at the single-cell level using next-generation sequencing (NGS). Authors Christina Fan, Ph.D., Glenn Fu, Ph.D., and Stephen Fodor, Ph.D., from Cellular Research, Inc., describe the technology and report results from several gene expression studies of cells from the human hematopoietic system.
Fan et al. note in the publication that single-cell analysis is increasingly necessary for understanding heterogeneous systems. Without this level of resolution, for example, large expression changes from a few cells appear the same as small expression changes from many cells.
In the manuscript, the scientists describe “a technically simple approach for gene expression cytometry” that uses cell- and molecule-specific barcodes to allow for the study of large numbers of genes in tens of thousands or even hundreds of thousands of cells at a time. Individual cells and primer-bearing beads are placed into microwells; when the cells are lysed, mRNAs hybridize to the barcoded primers on the beads. The beads are magnetically retrieved and moved to a tube for reverse transcription and amplification, followed by NGS analysis. “Sequencing of amplification products reveals the cell label, the molecular index, and the gene identity,” the authors write. “Computational analysis groups the reads based on the cell label and collapses the reads with the same molecular index and gene sequence into a single entry to correct for amplification bias, allowing the determination of absolute transcript numbers for each gene in each cell.”
“This unique labeling strategy and massively parallel approach make single-cell analysis of gene activity feasible and straightforward for any researcher with access to an NGS platform,” said Christina Fan, Ph.D., lead author on the paper and Staff Scientist at Cellular Research.
The publication also reports data from various types of hematopoietic cells using this technology. The scientists analyzed some 15,000 cells, estimating a consumable cost of pennies per cell when performed at high throughput. In a number of experiments, Fan et al. recapitulated decades of research results characterizing the individual cellular subtypes constituting an active hematopoietic system. They used other challenging biological problems to show the power of this new technology, demonstrating for example the detection of rare malignant cell types at very low numbers in a large background of normal cells.
“The Resolve technology will open vast new opportunities, and reveal new insights, in developmental biology and disease,” said Stephen Fodor, senior author on the paper and Chief Executive Officer of Cellular Research. “Examining the genetic profile of large populations of individual cells should enable the discovery of therapeutics with higher patient response rates and the development of more informative and earlier-stage diagnostics.”
The paper, entitled “Combinatorial labeling of single cells for gene expression cytometry,” appears in the journal Science.
Science 6 February 2015: Vol. 347 no. 6222 DOI: 10.1126/science.1258367
About the Authors
Stephen P.A. Fodor, Ph.D.
Dr. Fodor is Founder and Chief Executive Officer of Cellular Research, Inc. in Palo Alto, Calif. He received his B.S. in Biology and M.S. in Biochemistry from Washington State University and his Ph.D. in Chemistry from Princeton University. In the late 1980s, he and his colleagues were the first to invent and develop microarray technology culminating in a seminal 1991 publication in Science magazine. This work resulted in the founding of Affymetrix, where he led a handful of scientists in pioneering the fundamentals of array manufacturing, fluorescence instrument design, and assay development directed to scientific and commercial applications in the emerging field of genetic analysis and set the standards for the microarray industry in the early 1990s. The many uses of the microarray technology have included applications in mRNA expression profiling, mutation detection, genetic haplotype discovery, copy number analysis and the development of a variety of clinical products. Dr. Fodor managed the growth of this large commercial venture from inception, with oversight of numerous scientific projects and commercial products, as well as the marketing of several billion dollars of cumulative product sales. Dr. Fodor serves as a Trustee of the Carnegie Institution of Science and as a member of the National Academy of Engineering.
Glenn Fu, Ph.D.
Dr. Fu is Founder and Senior Scientist at Cellular Research, Inc. He received his B.A. in Molecular and Cell Biology from the University of California, Berkeley, and his Ph.D. in Molecular Epidemiology from the University of Michigan at Ann Arbor. He served as a post-doctoral fellow at the University of California, San Francisco working on the genetics of pediatric endocrinology. Dr. Fu was previously Director of Genotyping Research at Affymetrix. Before that, he held Director and Senior Director of Molecular Biology roles at Perlegen Sciences and Incyte Genomics. He worked closely with Dr. Fodor to invent and develop methods to label and count individual DNA molecules, which served as the core founding technology for Cellular Research.
Christina Fan, Ph.D.
Dr. Fan is Staff Scientist at Cellular Research, Inc. She received her Ph.D. in Bioengineering from Stanford University. As a graduate student at Stanford, she worked on the development of innovative genomic technologies and clinical applications of emerging genomic technologies. She has accumulated extensive experience in sequencing technologies, microfluidic device design, molecular biology assay development, and informatics analyses of large data sets. She has developed a sequencing-based noninvasive maternal blood test for fetal aneuploidy and a technique to determine the fetal genome from maternal blood. The former has become a commercially available diagnostic through Verinata Health, now part of Illumina. She has also developed microfluidic technologies for single cell analyses. Her research has been published in reputable journals including Nature, Cell, Nature Biotechnology, and PNAS. Her work has received recognitions including TR10 by MIT’s Technology Review. She was a recipient of Forbes