REVIEWS SINGLE-CELL OMICS Single-cell genome sequencing: current state of the science Charles Gawad1, Winston Koh2,3 and Stephen R. Quake2,3 Abstract | The field of single-cell genomics is advancing rapidly and is generating many new insights into complex biological systems, ranging from the diversity of microbial ecosystems to the genomics of human cancer. In this Review, we provide an overview of the current state of the field of single-cell genome sequencing. First, we focus on the technical challenges of making measurements that start from a single molecule of DNA, and then explore how some of these recent methodological advancements have enabled the discovery of unexpected new biology. Areas highlighted include the application of single-cell genomics to interrogate microbial dark matter and to evaluate the pathogenic roles of genetic mosaicism in multicellular organisms, with a focus on cancer. We then attempt to predict advances we expect to see in the next few years. Genetic mosaicism Cell theory provided an entirely new framework for In this Review, we describe the current state of the Occurs when there are at least understanding biology and disease by asserting that field, including approaches for cell isolation, whole-­ two genotypes in different cells cells are the basic unit of life1. The subsequent discov- genome amplification (WGA), DNA sequencing con- of the same organism. ery that DNA is the heritable programme that encodes siderations and sequence data analysis, and highlight Whole-genome the proteins that carry out cellular functions led to how recent progress is addressing some of the technical amplification the development of the fields of modern genetics and challenges associated with these approaches. We then The use of biochemical genomics2. Although bulk approaches for studying discuss how those advancements have begun to fulfil methods to produce multiple genetic variation have identified thousands of new some of the ambitious aspirations for the field in appli- copies of the entire genome. unicellular species and determined genetic aetiologies cations such as identifying new features of microbial for thousands of human diseases, most of that work ecosystems and characterizing human intercellular has been done at the level of the ecosystem or organ- genetic heterogeneity, in particular in cancer. ism3,4. However, we now know that diversity within an ecosystem of unicellular species is far greater than Technological challenges we can accurately measure by studying a mixed group Acquiring high-quality single-cell sequencing data has of organisms, and that the genomes within the cells of four primary technical challenges: efficient physical iso- an individual multicellular organism are not always lation of individual cells; amplification of the genome of the same. that single cell to acquire sufficient material for down- 1Departments of Oncology Single-cell genomics aims to provide new per- stream analyses; querying the genome in a cost-effective and Computational Biology, spectives to our understanding of genetics by bring- manner to identify variation that can test the hypotheses St. Jude Children’s Research ing the study of genomes to the cellular level (FIG. 1). of the study; and interpreting the data within the con- Hospital, Memphis, These tools are opening up new frontiers by dissect- text of biases and errors that are introduced during the Tennessee 38105, USA. 2Departments of ing the contributions of individual cells to the biology first three steps. To maximize the quality of single-cell Bioengineering and Applied of ecosystems and organisms. For example, it is now data and to ensure that the signal is separable from Physics, Stanford University, possible to use single-cell genomics to identify and technical noise, each of these variables requires careful Stanford, California 94304, assemble the genomes of unculturable microorgan- consideration when designing single-cell studies. USA. 5 genetic mosaicism 3Howard Hughes Medical isms , evalu­ate the roles of in normal 6 Institute, Stanford University, physiology and disease , and determine the contribu- Cell isolation. The first step in isolating individual California 94304, USA. tions of intra-tumour genetic heterogeneity in cancer cells from primary samples is to produce a suspension 7 Correspondence to S.R.Q. development or treatment response . However, this of viable single cells. This is not trivial when working [email protected] field rests on the ability to study a single DNA mol- with complex solid tissues, which require mechanical or doi:10.1038/nrg.2015.16 ecule from individually isolated cells, a process that is enzymatic dissociation that keeps the cells viable while Published online 25 Jan 2016 technically challenging. not biasing for specific subpopulations. In addition, NATURE REVIEWS | GENETICS VOLUME 17 | MARCH 2016 | 175 © 2016 Macmillan Publishers Limited. All rights reserved REVIEWS Example Output Unicellular Multicellular Output Example applications microorganisms eukaryotes applications List of variant frequencies Species Variant Allele frequency Cataloguing detected Sampling A W% variants and A Metagenomic their allele of entire B X% B frequencies in population C Y% C Tissue tissue samples D Z% D Variant Species A B C D Segregating detected Detection 1 variants to of rare A Mini- Single determine clonal microorganisms B metagenomic cell Cell 2 structures, E 3 identifying rare variants Genomic complexity of sample Phased genomic variants Assembly Genome Assembly of A C B D of new assemblies Single Single low-complexity microbial microorganism chromosome genomic regions genomes Chromosome 1a Chromosome 1b Figure 1 | Opportunities enabled by single-cell sequencing strategies. Single-cell approaches provide higher-resolution views of the genomic content of samples by reducing the complexity of the genomicNature Reviewssignal through | Genetics the physical separation of cells or chromosomes. Shown in the centre of the figure are schematics of the different levels of cellular complexity analysed by approaches in microorganisms and multicellular eukaryotes. Extending outwards are simplified diagrams of data outputs from these approaches and examples of applications, for unicellular microorganisms (extending to the left) and multicellular eukaryotes (extending to the right). For microbial genomics, decreasing the number of organisms enables the detection of rare microorganisms in a sample. Single-cell sequencing allows for the assembly of the entire genomes of new microorganisms. Single-cell sequencing of multicellular organisms can reveal rare genetic variants and provide information on the co‑occurrence of mutations and evolutionary history of samples. Single chromosome sequencing allows for the phasing of variants across a genome. diseased tissues can have different dissociation kinet- samples, depending on the environmental source ics when compared with their normal counterparts, as additional sample preparation and FACS setting con- well as varied dissociation between samples of the same siderations can also be required17. Finally, automated disease. Standard digestion protocols for commonly micromanipulation methods that use droplets or micro- studied tissues, as well as vigorous approaches for opti- mechanical valves in microfluidic devices are entering mizing the dissociation of rare or diseased tissues, are mainstream use18–20. Regardless of the method used, it areas that require further development. Laser-capture is also important to accurately confirm that a single cell microdissection8 provides a low-throughput way of has been physically isolated so that spurious biological isolating DNA from single cells in their native spatial conclusions are not made after evaluating chambers that context, but the quality of sequencing data derived from are empty or contain multiple cells. In the ideal case microdissected single cells has been relatively poor. this can be accomplished by obtaining microscopy data Finally, microfluidic and bead-based methods have from each chamber or well containing a single cell. been developed to specifically enrich for single circu- Various single-cell isolation technologies have lating tumour cells (CTCs), as reviewed in detail else- recently been reviewed, where the trade-offs in accuracy, where9. Environmental microbial samples also require throughput, reproducibility and ease of use were high- efficient lysis of bacteria, with requirements that can be lighted9,21,22. Most of the studies using these technolo- highly variable between species10. gies have been done to illustrate feasibility using a small Once in suspension, several approaches have been number of cells. Addressing many of the fundamental developed to isolate single cells. These include meth- biological questions that are uniquely approachable ods that require manual manipulation, such as serial with single-cell genomics will require the interrogation dilution11, micropipetting12, microwell dilution13 and of thousands of cells, making it more likely that tech- ­optical tweezers14. In addition, several protocols have nologies that are scalable through parallelization, such been developed to isolate intact cells or nuclei using as microfluidic-based approaches, are adopted for the Optical tweezers fluorescence-activated­ cell sorting (FACS)15. Nuclear long term. In addition, identifying scalable methods for Devices that use a laser to manipulate submicron isolation has the advantage of enabling single-­cell single-cell isolation