Multiomic Single Cell Sequencing Identifies Stemlike Nature of Mixed Phenotype Acute Leukemia and Provides Novel Risk Stratification.

Mixed phenotype acute leukemia (MPAL) is a leukemia whose biologic drivers are poorly understood, therapeutic strategy remains unclear, and prognosis is poor. We performed multiomic single cell (SC) profiling of 14 newly diagnosed adult MPAL patients to characterize the immunophenotypic, genetic, and transcriptional landscapes of MPAL. We show that neither genetic profile nor transcriptome reliably correlate with specific MPAL immunophenotypes. However, progressive acquisition of mutations is associated with increased expression of immunophenotypic markers of immaturity. Using SC transcriptional profiling, we find that MPAL blasts express a stem cell-like transcriptional profile distinct from other acute leukemias and indicative of high differentiation potential. Further, patients with the highest differentiation potential demonstrated inferior survival in our dataset. A gene set score, MPAL95, derived from genes highly enriched in this cohort, is applicable to bulk RNA sequencing data and was predictive of survival in an independent patient cohort, suggesting utility for clinical risk stratification.

Microfluidics-free single-cell genomics with templated emulsification.

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Bacterial Succession on Sinking Particles in the Ocean's Interior.

Sinking particles formed in the photic zone and moving vertically through the water column are a main mechanism for nutrient transport to the deep ocean, and a key component of the biological carbon pump. The particles appear to be processed by a microbial community substantially different from the surrounding waters. Single cell genomics and metagenomics were employed to describe the succession of dominant bacterial groups during particle processing. Sinking particles were extracted from sediment traps at Station Aloha in the North Pacific Subtropical Gyre (NPSG) during two different trap deployments conducted in July and August 2012. The microbial communities in poisoned vs. live sediment traps differed significantly from one another, consistent with prior observations by Fontanez et al. (2015). Partial genomes from these communities were sequenced from cells belonging to the genus Arcobacter (commensalists potentially associated with protists such as Radiolaria), and Vibrio campbellii (a group previously reported to be associated with crustacea). These bacteria were found in the particle-associated communities at specific depths in both trap deployments, presumably due to their specific host-associations. Partial genomes were also sequenced from cells belonging to Idiomarina and Kangiella that were enriched in live traps over a broad depth range, that represented a motile copiotroph and a putatively non-motile algicidal saprophyte, respectively. Planktonic bacterial cells most likely caught in the wake of the particles belonging to Actinomarina and the SAR11 clade were also sequenced. Our results suggest that similar groups of eukaryote-associated bacteria are consistently found on sinking particles at different times, and that particle remineralization involves specific, reproducible bacterial succession events in oligotrophic ocean waters.

Microbial community structure and function on sinking particles in the North Pacific Subtropical Gyre.

Sinking particles mediate the transport of carbon and energy to the deep-sea, yet the specific microbes associated with sedimenting particles in the ocean's interior remain largely uncharacterized. In this study, we used particle interceptor traps (PITs) to assess the nature of particle-associated microbial communities collected at a variety of depths in the North Pacific Subtropical Gyre. Comparative metagenomics was used to assess differences in microbial taxa and functional gene repertoires in PITs containing a preservative (poisoned traps) compared to preservative-free traps where growth was allowed to continue in situ (live traps). Live trap microbial communities shared taxonomic and functional similarities with bacteria previously reported to be enriched in dissolved organic matter (DOM) microcosms (e.g., Alteromonas and Methylophaga), in addition to other particle and eukaryote-associated bacteria (e.g., Flavobacteriales and Pseudoalteromonas). Poisoned trap microbial assemblages were enriched in Vibrio and Campylobacterales likely associated with eukaryotic surfaces and intestinal tracts as symbionts, pathogens, or saprophytes. The functional gene content of microbial assemblages in poisoned traps included a variety of genes involved in virulence, anaerobic metabolism, attachment to chitinaceaous surfaces, and chitin degradation. The presence of chitinaceaous surfaces was also accompanied by the co-existence of bacteria which encoded the capacity to attach to, transport and metabolize chitin and its derivatives. Distinctly different microbial assemblages predominated in live traps, which were largely represented by copiotrophs and eukaryote-associated bacterial communities. Predominant sediment trap-assocaited eukaryotic phyla included Dinoflagellata, Metazoa (mostly copepods), Protalveolata, Retaria, and Stramenopiles. These data indicate the central role of eukaryotic taxa in structuring sinking particle microbial assemblages, as well as the rapid responses of indigenous microbial species in the degradation of marine particulate organic matter (POM) in situ in the ocean's interior.

The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: a blueprint for thriving in and out of symbiosis.

BACKGROUND: Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum, and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions. To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced. RESULTS: Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.7 Mb), GC-rich (51%), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host. CONCLUSIONS: The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle.

Evidence for horizontal transmission from multilocus phylogeny of deep-sea mussel (Mytilidae) symbionts.

Many invertebrates at deep-sea hydrothermal vents depend upon bacterial symbionts for nutrition, and thus the mechanism of symbiont transmission, vertical (via the egg or sperm) or horizontal (from environment or contemporary hosts) is critically important. Under a strict maternal transmission model, symbiont and host mitochondrial genomes pass through the same individuals leading to congruent host-symbiont phylogenies. In contrast, horizontally transmitted symbionts are environmentally acquired, leading to incongruent host-symbiont phylogenies. Each of these transmission strategies is predicted to have different consequences for symbiont ecology and genome evolution. Deep-sea mussels (Bathymodiolinae) are globally distributed at deep-sea hydrothermal vents, depend upon chemoautotrophic symbionts for their survival, and are hypothesized to transmit their symbionts horizontally. This study explored bathymodioline symbiont ecology through quantification of symbionts at two hydrothermal vent sites and symbiont evolution using functional gene phylogenies. These phylogenies revealed a dramatically more complex evolutionary history than 16S ribosomal RNA phylogenies, suggesting that horizontal gene transfer may have played an important role in symbiont gene evolution. Tests of the strict maternal transmission hypothesis found that host-symbiont lineages were significantly decoupled across multiple genes. These findings expand our understanding of symbiont ecology and evolution, and provide the strongest evidence yet for horizontal transmission of bathymodioline symbionts.

Complete genome sequence of Candidatus Ruthia magnifica.

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Mollusca) is a member of the Vesicomyidae. Species within this family form symbioses with chemosynthetic Gammaproteobacteria. They exist in environments such as hydrothermal vents and cold seeps and have a rudimentary gut and feeding groove, indicating a large dependence on their endosymbionts for nutrition. The C. magnifica symbiont, Candidatus Ruthia magnifica, was the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced (Newton et al. 2007). Here we expand upon the original report and provide additional details complying with the emerging MIGS/MIMS standards. The complete genome exposed the genetic blueprint of the metabolic capabilities of the symbiont. Genes which were predicted to encode the proteins required for all the metabolic pathways typical of free-living chemoautotrophs were detected in the symbiont genome. These include major pathways including carbon fixation, sulfur oxidation, nitrogen assimilation, as well as amino acid and cofactor/vitamin biosynthesis. This genome sequence is invaluable in the study of these enigmatic associations and provides insights into the origin and evolution of autotrophic endosymbiosis.