Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

Multicellular organisms have co-evolved with complex consortia of viruses, bacteria, fungi and parasites, collectively referred to as the microbiota. In mammals, changes in the composition of the microbiota can influence a wide range of physiologic processes (including development, metabolism, and immune cell function) and are associated with susceptibility to multiple diseases. Alterations in the microbiota can also modulate host behaviors such as social activity, stress, and anxiety-related responses that are linked to diverse neuropsychiatric disorders. However, the mechanisms through which the microbiota influence neuronal activity and host behavior remain poorly defined. Here we demonstrate that manipulation of the microbiota in either antibiotictreated or germ-free adult mice results in significant deficits in fear extinction learning. Single nucleus RNA-sequencing of the medial prefrontal cortex of the brain revealed significant alterations in gene expression in multiple cell types including excitatory neurons and glial cells. Transcranial two-photon imaging following deliberate manipulation of the microbiota demonstrated that extinction learning deficits were associated with defective learning-related remodeling of postsynaptic dendritic spines and reduced activity in cue-encoding neurons in the medial prefrontal cortex. In addition to effects of manipulating the microbiota on behavior in adult mice, selective re-establishment of the microbiota revealed a limited neonatal developmental window in which microbiota-derived signals can restore normal extinction learning in adulthood. Lastly, unbiased metabolomic analysis identified four metabolites that were significantly downregulated in germ-free mice and were previous reported to be related to human and mouse models of neuropsychiatric disorders, suggesting that microbiota-derived compounds may directly affect brain function and behavior. Together, these data indicate that fear extinction learning requires microbiota-derived signals during both early postnatal neurodevelopment and in adult mice, with implications for our understanding of how diet, infection, and lifestyle influence brain health and subsequent susceptibility to neuropsychiatric disorders.

Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.

Summary Synonymous codon choices at the beginning of genes optimize 5′ RNA structures for enhanced translation initiation, but less is known about mechanisms that drive codon optimization downstream within the gene. To understand what determines codon choices across a gene, we generated 12,726 in situ codon mutants in the Escherichia coli essential gene infA and measured their fitness by combining multiplex automated genome engineering mutagenesis with amplicon deep sequencing (MAGE-seq). Correlating predicted 5′ RNA structure with fitness revealed that codons even far from the start of the gene are deleterious if they disrupt the native 5′ RNA conformation. These long-range structural interactions generate context-dependent rules that constrain codon choices beyond intrinsic codon preferences. Genome-wide RNA folding predictions confirm that natural codon choices far from the start codon are optimized in part to prevent disruption of native structures near the 5′ UTR. Our results shed light on natural codon distributions and should improve engineering of gene expression for synthetic biology applications.

The specific activation of Toll-like receptors (TLRs) has potential utility for a variety of therapeutic indications including antiviral immunotherapy and as vaccine adjuvants. TLR7 and TLR 8 may be activated by their native ligands, single-stranded RNA, or by small molecules of the imidazoquinoline family. However the use of TLR7/8 agonists for in vivo therapy is limited by instability, in the case of RNA, or systemic biodistribution and toxicity in the case of small molecule agonists. We hypothesized that unique lipid-like materials, termed "lipidoids," could be designed to efficiently deliver immunostimulatory RNA (isRNA) to TLR-expressing cells to drive innate and adaptive immune responses. A library of lipidoids was synthesized and screened for the ability to induce type I IFN activation in human peripheral blood mononuclear cells when combined with isRNA oligonucleotides. Effective lipidoid-isRNA nanoparticles, when tested in mice, stimulated strong IFN-α responses following subcutaneous injection, had robust antiviral activity that suppressed influenza virus replication, and enhanced antiovalbumin humoral and cell-mediated responses when used as a vaccine adjuvant. Further, we demonstrate that whereas all immunological activity was MyD88-dependent, certain materials were found to engage both TLR7-dependent and TLR7-independent activity in the mouse suggestive of cell-specific delivery. These lipidoid formulations, which are materials designed specifically for delivery of isRNA to Toll-like receptors, were superior to the commonly used N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate-RNA delivery system and may provide new tools for the manipulation of TLR responses in vitro and in vivo.innate immunity | dendritic cell | drug delivery | high-throughput screening