Mark Novotny

• Manager: Single Cell Lab at J. Craig Venter Institute-La Jolla

The ability to trace every cell in some model organisms has led to the fundamental understanding of development and cellular function. However, in plants the complexity of cell number, organ size and developmental time makes this a challenge even in the diminutive model plant Arabidopsis (Arabidopsis thaliana). Duckweed, basal non-grass aquatic mon...

Vaccination to prevent infectious disease is one of the most successful public health interventions ever developed. And yet, variability in individual vaccine effectiveness suggests that a better mechanistic understanding of vaccine-induced immune responses could improve vaccine design and efficacy. We have previously shown that protective antibody...

Single-cell genomics is rapidly advancing our knowledge of the diversity of cell phenotypes, both cell types and cell states. Driven by single-cell/nucleus RNA sequencing (scRNA-seq), comprehensive atlas projects covering a wide range of organisms and tissues are currently underway. As a result, it is critical that the transcriptional phenotypes di...

The ability to trace every cell in some model organisms has led to the fundamental understanding of development and cellular function. However, in plants the complexity of cell number, organ size and developmental times makes this a challenge even in the diminutive model plant Arabidopsis thaliana . Here we develop the Lesser Duckweed Lemna minuta...

Despite signs of infection—including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules—the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9...

Vaccination to prevent infectious disease is one of the most successful public health interventions ever developed. And yet, variability in individual vaccine effectiveness suggests a better mechanistic understanding of vaccine-induced immune responses could improve vaccine design and efficacy. We have previously shown that protective antibody leve...

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approac...

Single cell genomics is rapidly advancing our knowledge of cell phenotypic types and states. Driven by single cell/nucleus RNA sequencing (scRNA-seq) data, comprehensive atlas projects covering a wide range of organisms and tissues are currently underway. As a result, it is critical that the cell transcriptional phenotypes discovered are defined an...

Neutrophils are the most abundant white blood cells in the human body responsible for fighting viral, bacterial and fungi infections. Out of the 100 billion neutrophils produced daily, it is estimated that 10 % of these cells end up in oral biofluids. Because saliva is a fluid accessible through non-invasive techniques, it is an optimal source of c...

Neutrophils are the most abundant white blood cells in the human body responsible for fighting viral, bacterial and fungi infections. Out of the 100 billion neutrophils produced daily, it is estimated that 10 % of these cells end up in oral biofluids. Because saliva is a fluid accessible through non-invasive techniques, it is an optimal source of c...

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 iden...

Parallels among embryonic development, stem cells, and cancer have long been recognized. We identified, isolated, and characterized stem cells that first become committed to a mammary fate during embryogenesis; we refer to these cells as fetal mammary stem cells (fMaSCs). Lineage tracing, in vitro sphere formation, and in vivo transplantation studi...

[This corrects the article DOI: 10.1093/ve/vez020.].

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 iden...

The era of targeted therapies has seen significant improvements in depth of response, progression- free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches with...

The hadal zone remains one of the least studied environments because of its inaccessibility, in part because of hydrostatic pressures extending to 110 MPa. Few instruments are capable of sampling from such great depths. We have developed a full-ocean-depth-capable lander that can be fit with sampling packages for the collection of still images, vid...

Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the differe...

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with ana...

The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic,...

Table S1. Filtered Normalized Chromium Gene Expression Matrix, Related to Figures 1 and 2

Data S1. Scripts and Command Line Procedures, Related to STAR Methods and Figures 1 and 3

Table S3. NMF Cluster Designations and Signature Gene Lists, Related to Figures 1-3

Table S4. Expanded Gene Expression Matrix Incorporating Non-microfluidics samples, Related to Figure 5

In the version of this article initially published, NIH grant U01 MH106882 to F.H.G. was missing from the Acknowledgments. The error has been corrected in the HTML and PDF versions of the article.

Historically, enterovirus D68 (EV-D68) has primarily been associated with respiratory illnesses. However, in the summers of 2014 and 2016 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that the EV-D68 virus could be the causative agent of AFM during these recent outbreaks...

Vaccination as a strategy for the prevention of infectious disease has been one of the greatest success stories in modern medicine, resulting in the complete eradication of smallpox, the near eradication of polio, and the drastic lowering in the incidences of measles, mumps, influenza and other common diseases. In contrast to these remarkable succe...

Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single cell transcriptional profiling using RNA sequencing is producing "big data", enabling the identification of novel human cell types at an unprecedented rate. In this review, we summa...

Cells are fundamental functional units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single cell transcriptional profiling using RNA sequencing is producing “big data”, enabling the identification of novel human cell types at an unprecedented rate. In this review, we sum...

Background: A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states a...

We describe convergent evidence from transcriptomics, morphology and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a novel group of human interneurons wit...

Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack (acute myocardial infarction, AMI), there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque ruptu...

Candidate phyla (CP) are broad phylogenetic clusters of organisms that lack cultured representatives. Included in this fraction is the candidate Parcubacteria superphylum. Specific characteristics that have been ascribed to the Parcubacteria include reduced genome size, limited metabolic potential, and exclusive reliance on fermentation for energy...

Nature Communications 7 : Article number: 11022 10.1038/ncomms11022 ( 2016 ); Published 19 April 2016 ; Updated 17 March 2017 An incorrect version of Supplementary Data 1, in which normalized counts were analysed instead of raw counts, resulting in a smaller number of differentially expressed genes, was inadvertently published with this article.

Next generation sequencing of the RNA content of single cells or single nuclei (sc/nRNA-seq) has become a powerful approach to understand the cellular complexity and diversity of multicellular organisms and environmental ecosystems. However, the fact that the procedure begins with a relatively small amount of starting material, thereby pushing the...

The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are compose...

Nature Communications 7: Article number:1102210.1038/ncomms11022 (2016); Published April192016; Updated June142016 In the original version of this Article, the middle names or initials of the authors Suguna Rani Krishnaswami, Jerika J. Barron, Martijn J.E. Kelder, Sarah L. Parylak, Apua C.M. Paquola and Jennifer A. Erwin were omitted from the auth...

Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through eval...

Supplementary Figures 1-4 and Supplementary Table 1

Supplementary dataset 1

Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack (acute myocardial infarction, AMI), there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque ruptu...

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described i...

Hadal ecosystems are found below 6,000 m depth, occupying 1-2% of the total area of the ocean. Microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived f...

Significance Development of preimplantation embryos entails global DNA demethylation on the zygotic genome. The original thought was that TET-deficient embryos would be unlikely to survive early embryogenesis because they would be unable to mediate genome-wide demethylation in the zygote and preimplantation embryo. However, mice lacking the individ...

Significance One of the central goals of developmental biology and medicine is to ascertain the relationships between the genotype and phenotype of cells. Single-cell transcriptome analysis represents a powerful strategy to reach this goal. We advance these strategies to single nuclei from neural progenitor cells and dentate gyrus tissue, from whic...

Significance This research highlights the discovery and genome reconstruction of a member of the globally distributed yet uncultivated candidate phylum TM6 (designated TM6SC1). In addition to the 16S rRNA gene, no other genomic information is available for this cosmopolitan phylum. This report also introduces a mini-metagenomic approach based on th...

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation (1-3). Viruses, which are ubiquitous and the most numerous entities on our planet (4) and important in all environments (5), have yet to be revealed via similar approaches. Here we describe an approach for isolating a...

Sequences were screened for chimeras by the submitter using Decipher 2013. ##Assembly-Data-START## Assembly Method :: Spades v. 2012; CLC v. 5.1 Coverage :: 10 Sequencing Technology :: Illumina ##Assembly-Data-END##

Although biofilms have been shown to be reservoirs of pathogens, our knowledge of the microbial diversity in biofilms within critical areas, such as health care facilities, is limited. Available methods for pathogen identification and strain typing have some inherent restrictions. In particular, culturing will yield only a fraction of the species p...

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome...

The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such...

SAR86 denotes a 16S clade of gammaproteobacteria that are ubiquitous in ocean surface waters. So far, SAR86 is resistant to cultivation; thus, little is known about the genome contents or physiology of this clade. Recently, four partial genome sequences for SAR86 subclades I and II were published. Here, we present the draft genome sequence of a sin...

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition(1,2). There are examples where many genes need to be deleted to unmask hidden gene functions(3,4). Despite the potential for important discoveries, genetic interactions involving three or more genes are la...

Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard...

Bacteria in the 16S rRNA clade SAR86 are among the most abundant uncultivated constituents of microbial assemblages in the surface ocean for which little genomic information is currently available. Bioinformatic techniques were used to assemble two nearly complete genomes from marine metagenomes and single-cell sequencing provided two more partial...

Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of DNA from single cells of bacteria that cannot be cultured. Assembling a genome is challenging, however, because MDA generates highly nonuniform coverage of the genome. Here we describe an algorithm tailored for short-read data from single cells t...

Abundance of the functional OG category Signal Transduction (T) for deep ocean metagenomes compared to the Sargasso Sea metagenomes. (PDF)

Statistical hypothesis testing implemented in the program STAMP [34] for differentially abundant orthologous groups (OGs) between the PRT metagenome and the Sargasso Sea (A) GS00c and (B) GS00d metagenomes. Results are shown for the Fisher's exact test using the Newcombe-Wilson method for calculating confidence intervals (CIs) at the 95% nominal co...

Fragment recruitment coverage plots for the unassembled nonredundant PRT metagenomic reads against the seven fully sequenced fosmids from Martín-Cuadrado et al. [49]. (A) KM3-26-C03 (NCBI Accession number: GU058051), (B) KM3-28-H12 (GU058052), (C) KM3-29-C02 (GU058053), (D) KM3-41-E12 (GU058054), (E) KM3-45-H11 (GU058057), (F) KM3-54-A05 (GU058055)...

Relative abundance of assignable COG categories and distribution within phylum-level (and class-level for the Proteobacteria) groupings based on APIS. Only phyla contributing ≥0.2% of the total proteins classified are shown. COG categories are as follows: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production...

Phylogenetic trees depicting the relationship of the 16S rRNA gene sequences for the (A) Alphaproteobacterium Rhodospirillales bacterium JCVI-SC AAA001, (B) Gammaproteobacterium Oceanospirillales bacterium JCVI-SC AAA002, (C) Bacteroidetes Flavobacteriales bacterium JCVI-SC AAA003, and (D) Planctomycetes bacterium JCVI-SC AAA004. The rRNA gene sequ...

Abundance of the functional OG category Inorganic ion transport and metabolism (P) for deep ocean metagenomes compared to the Sargasso Sea metagenomes. (PDF)

Chemical and biological constituents of hadal (6,000 m) seawater. Data previously published in Eloe et al. [16]. (DOC)

Detailed assembly and putative contaminant statistics for single-cell genomes. Mate pair ratio represents the total number of mated reads divided by the total number of reads. % Unique designates the percentage of reads after exclusion of duplicate reads, homopolymers, and removing N's. Clean datasets consisted of removal of all contigs less than 1...

Preparation of DNA for 454 pyrosequencing. (DOC)

Comparison of the unassembled nonredundant PRT reads against the DeepMed [5]; a 7-depth profile from Station ALOHA (10 m, 70 m, 130 m, 200 m, 500 m, 770 m, 4,000 m) [7]; the Mediterranean deep chlorophyll maximum (DCM) [48]; 1,300 m depth sediment and 1,000 m depth water column from the Sea of Marmara [79]; black smoker chimney in the Mothra hydrot...

Abundance of the functional OG category Transcription (K) for deep ocean metagenomes compared to the Sargasso Sea metagenomes. (PDF)

(A) Significantly different transporter classifications (TC IDs) identified between the deep and shallow metagenome comparisons. Highlighted in blue are the TC IDs that were differentially over-represented in the deep metagenomes. (B) Transporter family abundances for the five metagenomes. (XLS)

The paucity of sequence data from pelagic deep-ocean microbial assemblages has severely restricted molecular exploration of the largest biome on Earth. In this study, an analysis is presented of a large-scale 454-pyrosequencing metagenomic dataset from a hadopelagic environment from 6,000 m depth within the Puerto Rico Trench (PRT). A total of 145...

Primers specific for phages T4 and lambda loci used in multiplex PCR to identify phage isolated. (PDF)

Reference mapping statistics (all sequence lengths are given in bp). (PDF)

Statistics following SVG methodology on 16 test samples. CLSM numbers corresponds to viruses detected during microscopy, MDA refers to a positive (+) or negative (−) when amplification was detected by gel electrophoresis of wells containing viral particles. A positive specific PCR is denoted by the genotype obtained after multiplex PCR of the ampli...

Allele variation analysis using CLC Genomics Workbench. (PDF)

BLAST analysis of unmapped read sequences following reference mapping. (PDF)

De novo assembly statistics. (PDF)

Contaminants found in 16S PCR analysis of MDA reactions. (PDF)

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new appro...

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of singl...

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for lo...

A 768-lane DNA sequencing system based on micromachined plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented in large-format (25 cm by 50 cm) microfabricated devices with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read...

We outline some of the most important scientific problems that were overcome in this multiyear project. These include (a) the high-yield microfabrication of zero-defect bonded glass plates at sizes greatly exceeding semiconductor fabrication, (b) the integration of electrodes, reservoirs, and high-pressure seals into these plates, (c) detector opti...

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highl...