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.

Therapeutic proteins and antibodies represent a $125 billion annual market. Chinese Hamster Ovary (CHO) derived cell lines are the preferred host cells for the production of therapeutic proteins. Here, we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45Gb genomic sequence with 24,383 predicted genes. We associate most scaffolds to 21 microfluidically-isolated chromosomes to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affects therapeutic protein quality, and viral susceptibility genes, which affect cell engineering and regulatory concerns. Specifically, homologs for most human glycosylation-associated genes are identified in the CHO-K1 genome, although 141 are not expressed under exponential growth. In addition, many important viral entry genes are present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We demonstrate how the availability of this genome sequence may facilitate genome-scale science for biopharmaceutical protein production.

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing with no requirement for a quiescent stem cell sub-population. By means of double–labeling RNA in situ hybridization, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self renewal.

Many cancers have substantial genomic heterogeneity within a given tumor, and to fully understand that diversity requires the ability to perform single cell analysis. We performed targeted sequencing of a panel of single nucleotide variants (SNVs), deletions, and IgH sequences in 1,479 single tumor cells from six acute lymphoblastic leukemia (ALL) patients. By accurately segregating groups of cooccurring mutations into distinct clonal populations, we identified codominant clones in the majority of patients. Evaluation of intraclonal mutation patterns identified clone-specific punctuated cytosine mutagenesis events, showed that most structural variants are acquired before SNVs, determined that KRAS mutations occur late in disease development but are not sufficient for clonal dominance, and identified clones within the same patient that are arrested at varied stages in B-cell development. Taken together, these data order the sequence of genetic events that underlie childhood ALL and provide a framework for understanding the development of the disease at single-cell resolution.single-cell genomics | acute lymphoblastic leukemia | intratumor heterogeneity | clonal evolution | cytosine mutagenesis A more comprehensive understanding of how malignancies develop could facilitate the rational development of novel anticancer treatment and prevention strategies. Large projects that aim to comprehensively characterize somatic mutations in cancer samples have cataloged many of the recurrent genomic lesions in a wide variety of tumors (1). However, these studies do not measure the correlated cooccurrence of genomic lesions between different cells, which is required for understanding the clonal structure of a tumor as well as for rigorously determining temporal ordering of mutation acquisition. Other studies have provided some temporal resolution of mutation segregation patterns from diagnosis to disease recurrence, allowing for post hoc inference of intratumor clonal heterogeneity at diagnosis (2-5). However, approaches that rely on mutant allele frequencies to determine clonal structure require multiple samples from the same patient and are unable to resolve clones with mutations present at similar frequencies, which is a prerequisite to unambiguously determine the clonal structure and delineate the evolution of the disease (3-5). In principle, single cell genomics provides the most rigorous method to determine the clonal heterogeneity of tumors; as discussed below, there have been recent advances in this approach, but technical limitations have until now prevented it from fully addressing the questions of interest.Studies of pediatric acute lymphoblastic leukemias (ALL) have provided a limited ordering of the genetic events that underlie childhood leukemogenesis by studying prediagnostic samples. For example, ETV6 -RUNX1 translocations, which occur in about a third of patients under 10 y of age, have been shown to occur in utero by tracking the translocation back to neonatal blood spots (6, 7). In addition, a recent report ...

Significance Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape of circulating RNA in human subjects. By focusing on tissue-specific genes, we were able to identify the relative contributions of these tissues to circulating RNA and monitor changes during tissue development and neurodegenerative disease states.

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration.DOI: http://dx.doi.org/10.7554/eLife.00569.001

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.