76 Citations
The Maternal-to-Zygotic transition MZT is a reprograming process encompassing zygotic genome activation ZGA and the clearance of maternally-provided mRNAs While some factors regulating MZT have been identified there are thousands of maternal RNAs whose function has not been ascribed yet Here we have performed a proof-of-principle CRISPR-RfxCas d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT Bckdk mRNA knockdown caused epiboly defects ZGA deregulation H K ac reduction and a partial impairment of miR- processing Phospho-proteomic analysis revealed that Phf Baf a a chromatin remodeling factor is ... More
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. Less
Genetic studies have identified thousands of individual disease-associated non-coding alleles but identification of the causal alleles and their functions remain critical bottlenecks Even though CRISPR-Cas editing has enabled targeted modification of DNA inefficient editing leads to heterogeneous outcomes across individual cells limiting the ability to detect functional consequences of disease alleles To overcome these challenges we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits assays the transcriptome and measures cell surface protein expression We apply this approach to investigate the effects of gene disruption deletions in regulatory regions and non-coding single nucleotide polymorphisms We identify ... More
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells. Less
Mobile phones contaminated with pathogenic microorganisms have the potential to act as trojan horses The microbial signatures present on their surfaces most probably vary across different geographical regions As a result mobile phones belonging to international conference attendees may serve as a model for global microbial dissemination posing potential risks to public health and biosecurity This study aimed to profile the microbes present on mobile phones belonging to delegates attending an international scientific conference through use of metagenomic shotgun DNA sequencing Results A total of microbial hits were accumulated across mobile phones inclusive of bacteria viruses fungi and protozoa Of ... More
Mobile phones, contaminated with pathogenic microorganisms, have
the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to
international conference attendees may serve as a model for global microbial dissemination,
posing potential risks to public health and biosecurity. This study aimed to profile the microbes present on mobile phones belonging to delegates
attending an international scientific conference through use of metagenomic shotgun DNA
sequencing. Results: A total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of
882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human
pathogens, including ESKAPE and HACEK bacteria were found on mobile phones Less
the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to
international conference attendees may serve as a model for global microbial dissemination,
posing potential risks to public health and biosecurity. This study aimed to profile the microbes present on mobile phones belonging to delegates
attending an international scientific conference through use of metagenomic shotgun DNA
sequencing. Results: A total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of
882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human
pathogens, including ESKAPE and HACEK bacteria were found on mobile phones Less
The Olink Explore platform enables high-throughput protein biomarker discovery through Proximity Extension Assay PEA technology combined with Next Generation Sequencing NGS on Illumina instruments This approach allows for the simultaneous measurement of thousands of human plasma proteins with minimal sample volumes The Explore library offers approximately protein assays while the smaller Explore -plex panels cater to more targeted studies The platform excels in detecting low-abundance proteins such as cytokines and chemokines and is particularly effective for challenging sample types like cerebrospinal fluid CSF where protein content is typically low In this chapter we emphasize critical dry-lab considerations including CSF handling ... More
The Olink® Explore platform enables high-throughput protein biomarker discovery through Proximity Extension Assay (PEA) technology combined with Next Generation Sequencing (NGS) on Illumina instruments. This approach allows for the simultaneous measurement of thousands of human plasma proteins with minimal sample volumes. The Explore 3072 library offers approximately 3000 protein assays, while the smaller Explore 384-plex panels cater to more targeted studies. The platform excels in detecting low-abundance proteins, such as cytokines and chemokines, and is particularly effective for challenging sample types like cerebrospinal fluid (CSF), where protein content is typically low. In this chapter, we emphasize critical dry-lab considerations, including CSF handling, study design, sample size determination, instrumentation requirements, and post-experiment data management. Proper planning and execution of these factors are essential for optimizing performance and ensuring reliable outcomes when using Olink®'s platform. Less
While contemporary short-read single cell RNA-sequencing allows to decipher tissue composition discrimination between transcript isoforms remains challenging Here we propose single cell long-read isoform sequencing scLIS-seq and highlight its performance on Jurkat and HEK T cells in direct comparison to Smart-seq xpress SS X scLIS-seq demonstrates sensitive gene and transcript detection with high correlation compared to SS X and detects at least isoforms of over genes while of the reads supported novel isoforms Direct comparison of the scLIS-seq isoforms to SS X-reconstructed isoforms demonstrated scLIS-seq s superiority Overall scLIS-seq provides a powerful scRNA-seq strategy enabling long-read transcriptome analysis and isoform ... More
While contemporary short-read single cell RNA-sequencing allows to decipher tissue composition, discrimination between transcript isoforms remains challenging. Here, we propose single cell long-read isoform sequencing (scLIS-seq), and highlight its performance on Jurkat and HEK293T cells in direct comparison to Smart-seq3xpress (SS3X). scLIS-seq demonstrates sensitive gene and transcript detection with high correlation compared to SS3X and detects at least 10 isoforms of over 2600 genes, while 17.1–21.6% of the reads supported novel isoforms. Direct comparison of the scLIS-seq isoforms to SS3X-reconstructed isoforms demonstrated scLIS-seq’s superiority. Overall, scLIS-seq provides a powerful scRNA-seq strategy, enabling long-read transcriptome analysis and isoform detection. Less
Single-cell transcriptomics is a key tool for unravelling metabolism and tissue diversity in model organisms Its potential for elucidating the ecological roles of microeukaryotes especially non-model ones remains largely unexplored This study employed the Smart-seq protocol on Ochromonas triangulata a microeukaryote lacking a reference genome showcasing how transcriptional states align with two distinct growth phases a fast-growing phase and a slow-growing phase Besides the two expected expression clusters each corresponding to either growth phase a third transcriptional state was identified across both growth phases Metabolic mapping revealed a boost of photosynthetic activity in the fast growth over the slow growth ... More
Single-cell transcriptomics is a key tool for unravelling metabolism and tissue diversity in model organisms. Its potential for elucidating the ecological roles of microeukaryotes, especially non-model ones, remains largely unexplored. This study employed the Smart-seq2 protocol on Ochromonas triangulata, a microeukaryote lacking a reference genome, showcasing how transcriptional states align with two distinct growth phases: a fast-growing phase and a slow-growing phase. Besides the two expected expression clusters, each corresponding to either growth phase, a third transcriptional state was identified across both growth phases. Metabolic mapping revealed a boost of photosynthetic activity in the fast growth over the slow growth stage, as well as down-regulation trend in pathways associated with ribosome functioning, CO2 fixation, and carbohydrate catabolism characteristic of the third transcriptional state. In addition, carry-over rRNA reads recapitulated the taxonomic identity of the target while revealing distinct bacterial communities, in co-culture with the eukaryote, each associated with distinct transcriptional states. This study underscores single-cell transcriptomics as a powerful tool for characterizing metabolic states in microeukaryotes without a reference genome, offering insights into unknown physiological states and individual-level interactions with different bacterial taxa. This approach holds broad applicability to describe the ecological roles of environmental microeukaryotes, culture-free and reference-free, surpassing alternative methods like metagenomics or metatranscriptomics. Less
The local arrangement of microbes can profoundly impact community assembly function and stability However our understanding of the spatial organization of the human gut microbiome at the micron scale is limited Here we describe a high-throughput and streamlined method called Split-And-pool Metagenomic Plot-sampling sequencing SAMPL-seq to capture spatial co-localization in a complex microbial consortium The method obtains microbial composition of micron-scale subcommunities through split-and-pool barcoding SAMPL-seq analysis of the healthy human gut microbiome identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals These co-localized microbes organize into spatially distinct groups or spatial hubs dominated by ... More
The local arrangement of microbes can profoundly impact community assembly, function and stability. However, our understanding of the spatial organization of the human gut microbiome at the micron scale is limited. Here we describe a high-throughput and streamlined method called Split-And-pool Metagenomic Plot-sampling sequencing (SAMPL-seq) to capture spatial co-localization in a complex microbial consortium. The method obtains microbial composition of micron-scale subcommunities through split-and-pool barcoding. SAMPL-seq analysis of the healthy human gut microbiome identified bacterial taxa pairs that consistently co-occurred both over time and across multiple individuals. These co-localized microbes organize into spatially distinct groups or ‘spatial hubs’ dominated by Bacteroidaceae, Ruminococcaceae and Lachnospiraceae families. Using inulin as a dietary perturbation, we observed reversible spatial rearrangement of the gut microbiome where specific taxa form new local partnerships. Spatial metagenomics using SAMPL-seq can unlock insights into microbiomes at the micron scale. Less
T cells are key players in adaptive immunity The specificity of T cells is determined by the sequences of the hypervariable T cell receptor TCR and chains Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling it does not provide chain pairings which are essential for determining T cell specificity In contrast single-cell TCR sequencing technologies produce paired chain data but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies Here we present TIRTL-seq Throughput-Intensive Rapid TCR Library sequencing a novel approach that generates ready-to-sequence TCR libraries from live cells in ... More
ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire. Less
We developed an automated high-throughput Smart-seq HT Smart-seq workflow that integrates best practices and an optimized protocol to enhance efficiency scalability and method reproducibility This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity In a rigorous comparison with the X platform using human primary CD T-cells HT Smart-seq demonstrated higher cell capture efficiency greater gene detection sensitivity and lower dropout rates Additionally when sufficiently scaled HT Smart-seq achieved a comparable resolution of cellular heterogeneity to X Notably through T-cell receptor TCR reconstruction HT Smart-seq identified a greater number of productive alpha and beta chain ... More
We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples. Less
Primordial follicle activation PFA is a pivotal event in female reproductive biology coordinating the transition from quiescent to growing follicles This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells GCs and oocytes with the progression of the PFA process Wntless Wls conditional knockout cKO mice served as a unique model suppressing the transition from pre-GCs to GCs and disrupting somatic cell-derived WNT signaling in the ovary Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice leading to a divergent trajectory while ... More
Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis. Less
Menstrual toxic shock syndrome mTSS is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin- TSST- superantigen Herein we screened a library of small bioactive molecules for the ability to inhibit transcription of the TSST- gene without inhibiting the growth of S aureus The dominant positive regulator of TSST- is the SaeRS two-component system TCS and we identified phenazopyridine hydrochloride PP-HCl that repressed the production of TSST- by inhibiting the kinase function of SaeS PP-HCl competed with ATP for ... More
Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting the growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed the production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets the virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS. Less
Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions ligating an adapter to create a circular molecule sequencing the circular molecule to obtain sequence reads generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample
Corticospinal neurons CSNs synapse directly on spinal neurons a diverse assortment of cells with unique structural and functional properties necessary for body movements CSNs modulating forelimb behavior fractionate into caudal forelimb area CFA and rostral forelimb area RFA motor cortical populations Despite their prominence the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted Here we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback CFA and RFA CSNs target similar spinal neuron types ... More
Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse assortment of cells with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted. Here, we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal neuron types, with notable exceptions that suggest that these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive highly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior. Less
Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy Correct estimation of the ploidy of single-cell genomes is paramount for downstream analysis Based only on single-cell DNA sequencing information scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status including whole-genome duplications We demonstrate scAbsolute s capabilities using experimental cell multiplets a FUCCI cell cycle expression system and a benchmark against state-of-the-art methods scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and has the potential to enable improvements in a number of downstream analyses
Hematopoietic stem cells HSCs develop from the hemogenic endothelium HE in the aorta- gonads-and mesonephros AGM region and reside within Intra-aortic hematopoietic clusters IAHC along with hematopoietic progenitors HPC The signalling mechanisms that distinguish HSCs from HPCs are unknown Notch signaling is essential for arterial specification IAHC formation and HSC activity but current studies on how Notch segregates these different fates are inconsistent We now demonstrate that Notch activity is highest in a subset of GFI HSC-primed HE cells and is gradually lost with HSC maturation We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH ... More
Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta. Less
Memory encodes past experiences thereby enabling future plans The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks These transcriptional signatures implicate neuropeptide and BDNF signalling MAPK and CREB activation ubiquitination pathways and synaptic connectivity as key components of long-term memory Notably upon long-term memory ... More
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory. Less
In the fields of human health and agricultural research low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples However a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature In this study we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits the KAPA HyperPlus kit Roche the DNA Prep kit Illumina and an IDT kit using human DNA samples Metrics evaluated included imputation concordance with ... More
In the fields of human health and agricultural research, low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples. However, a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature. In this study, we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits—the KAPA HyperPlus kit (Roche), the DNA Prep kit (Illumina), and an IDT kit—using 96 human DNA samples. Metrics evaluated included imputation concordance with high-depth genotypes, coverage, duplication rates, time for library preparation, and additional optimization requirements. Despite slightly elevated duplication rates in IDT kits, we find that all four kits perform well in terms of imputation accuracy, with IDT kits being only marginally less performant than Illumina and Roche kits. Laboratory handling of the kits was similar: thus, the choice of a kit will largely depend on (1) existing or planned infrastructure, such as liquid handling capabilities, (2) whether a specific characteristic is desired, such as the use of full-length adapters, shorter processing times, or (3) use case, for instance, long vs short read sequencing. Our findings offer a comprehensive resource for both commercial and research workflows of low-cost library preparation methods suitable for high-throughput low coverage whole genome sequencing. Less
Wastewater-based SARS-CoV- epidemiology WBE has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants However for a timely transmission of results sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis comparative quality control of the analytical procedures is essential to report reliable and comparable results In this study we performed an interlaboratory comparison at laboratories ... More
Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results.In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values.Despite the different procedures, a high concordance regarding the SARS-CoV-2 PCR quantification could be achieved with low variation between the workflows. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/mL wastewater and had limited predictive value when analyzing at a genome coverage below 60%.This interlaboratory test demonstrates that despite different extraction and analysis methods, a high agreement of the SARS-CoV-2 genome copy equivalents could be achieved. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection. Less
Next-generation sequencing NGS technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research Skim sequencing which surveys the entire genome at low coverage has become feasible for quantitative trait locus QTL mapping and genomic selection in various crops However the genome complexity of allopolyploid crops such as wheat Triticum aestivum L still poses a significant challenge for genome-wide genotyping Targeted sequencing of the protein-coding regions i e exome reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping We developed a method called skim exome capture SEC ... More
Next-generation sequencing (NGS) technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research. Skim sequencing, which surveys the entire genome at low coverage, has become feasible for quantitative trait locus (QTL) mapping and genomic selection in various crops. However, the genome complexity of allopolyploid crops such as wheat (Triticum aestivum L.) still poses a significant challenge for genome-wide genotyping. Targeted sequencing of the protein-coding regions (i.e., exome) reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping. We developed a method called skim exome capture (SEC) that combines the strengths of these existing technologies and produces targeted genotyping data while decreasing the cost on a per-sample basis compared to traditional exome capture. Specifically, we fragmented genomic DNA using a tagmentation approach, then enriched those fragments for the low-copy genic portion of the genome using commercial wheat exome baits and multiplexed the sequencing at different levels to achieve desired coverage. We demonstrated that for a library of 48 samples, ∼7–8× target coverage was sufficient for high-quality variant detection. For higher multiplexing levels of 528 and 1056 samples per library, we achieved an average coverage of 0.76× and 0.32×, respectively. Combining these lower coverage SEC sequencing data with genotype imputation using a customized wheat practical haplotype graph database that we developed, we identified hundreds of thousands of high-quality genic variants across the genome. The SEC method can be used for high-resolution QTL mapping, genome-wide association studies, genomic selection, and other downstream applications. Less
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded ss DNA We utilized a yeast model to explore mutagenesis by glycidamide a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines The most frequent mutations in adenines occurred in the nAt nGt trinucleotide motif Base substitutions A G in this motif relied on Rev translesion polymerase activity Inactivating Rev did not alter the nAt trinucleotide preference suggesting it may be an intrinsic specificity of the chemical reaction ... More
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents. Less
The gut microbiome is complex raising questions about the role of individual strains in the community Here we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid -dehydroxylation niche Omitting Clostridium scindens Cs and Clostridium hylemonae Ch eliminates secondary bile acid production and reshapes the community in a highly specific manner eight strains change in relative abundance by -fold In single-strain dropout communities Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent However Clostridium sporogenes increases -fold in the Cs but ... More
The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Less
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy Here we introduce scEC T-seq s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells In this protocol a single cell ... More
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival. A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity. Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy. Here, we introduce scEC&T-seq ( s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells. In this protocol, a single cell’s DNA is separated from its polyadenylated RNA as described by Macaulay et al. (2015) 1 . This is followed by removal of linear DNA through exonuclease digestion and further enrichment of circular DNA by rolling circle amplification with φ29 polymerase 2-4 . The separated mRNA from the same cell is processed using on-bead Smart-seq2 1 . The duration of the entire procedure from cell sorting to library preparation is approximately 8 days. Our scEC&T-seq protocol has been validated in single cancer cells from neuroblastoma cell lines and primary tumors, and in normal single T-cells isolated from patient’s blood. Besides identifying large, oncogene-containing circular DNAs in cancer cells, our method also captures other smaller circular DNAs, which have been previously described in both cancer and non-malignant cells 5 . We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development. Less
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development however the molecular mechanisms underlying this phenomenon in mammals remain poorly described Here we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days and and compared the species using a framework for time-resolved single-cell differentiation-flows analysis We find convergence toward similar cell-state compositions at E supported by the quantitatively conserved expression of transcription factors despite divergence in surrounding trophoblast and hypoblast signaling However we ... More
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals. Less
Despite advances in high-dimensional cellular analysis the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways
The microbial guild coupling anammox and nitrite nitrate-dependent anaerobic methane oxidation n-DAMO is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater Here metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater respectively We find that different nitrogen loadings i e vs kg N m d lead to different combinations of anammox bacteria and anaerobic methanotrophs Candidatus Methanoperedens and Candidatus Methylomirabilis which play primary roles for ... More
The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs (“Candidatus Methanoperedens” and “Candidatus Methylomirabilis”), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to “Ca. Methylomirabilis” are recovered, while one surprisingly expresses nitrate reductases, which we designate as “Ca. Methylomirabilis nitratireducens” representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of “Ca. Methylomirabilis” bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea. Less
Described herein are methods for stratifying and evaluating melanoma treatment response in a subject using single-cell RNA sequencing scRNA-seq and a two-step deconvolution analysis and optionally administering a treatment depending on the results Embodiment described herein are methods for stratifying and evaluating melanoma treatment response in a subject based on single cell or bulk RNA sequencing bulk transcriptome profiling and or transcript counting and a two-step deconvolution analysis and optionally administering a treatment depending on the results
Background With an increasing interest in the manipulation of methane produced from livestock cultivation the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with low-methane emitters Previously marsupial species were shown to be enriched for novel lineages of Methanocorpusculum as well as Methanobrevibacter Methanosphaera and Methanomassiliicoccales Despite sporadic reports of Methanocorpusculum from stool samples of various animal species there remains little information on the impacts of these methanogens on their hosts Results Here we characterise novel host-associated species of Methanocorpusculum to explore unique host-specific genetic factors and their associated metabolic potential We performed comparative analyses on Methanocorpusculum ... More
Background
With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.
Results
Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).
Conclusions
Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated. Less
With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with ‘low-methane’ emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts.
Results
Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.).
Conclusions
Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated. Less
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells NSPCs that give rise to the neurons oligodendrocytes and astrocytes of the mature brain Functional study of these cell types has been hampered by a lack of precise purification methods We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers CD THY lo cells were enriched for radial glia which robustly engrafted and differentiated into all three neural lineages in the mouse brain THY hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial ... More
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24−THY1−/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1−/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA− bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment. Less
Breastmilk is thought to influence the infant gut by supplying prebiotics in the form of human milk oligosaccharides and potentially seeding the gut with breastmilk microbes However the presence of a breastmilk microbiota and origins of these microbes are still debated As a pilot study we assessed the microbes present in expressed breastmilk at six-weeks postpartum using shotgun metagenomic sequencing in a heterogenous cohort of women who delivered by vaginal n and caesarean delivery n In addition we estimated the microbial load of breastmilk at six-weeks post-partum with quantitative PCR targeting the S rRNA gene Breastmilk at six-weeks postpartum had ... More
Breastmilk is thought to influence the infant gut by supplying prebiotics in the form of human milk oligosaccharides and potentially seeding the gut with breastmilk microbes. However, the presence of a breastmilk microbiota and origins of these microbes are still debated. As a pilot study, we assessed the microbes present in expressed breastmilk at six-weeks postpartum using shotgun metagenomic sequencing in a heterogenous cohort of women who delivered by vaginal (n = 8) and caesarean delivery (n = 8). In addition, we estimated the microbial load of breastmilk at six-weeks post-partum with quantitative PCR targeting the 16S rRNA gene. Breastmilk at six-weeks postpartum had a low microbial mass, comparable with PCR no-template and extraction controls. Microbes identified through metagenomic sequencing were largely consistent with skin and oral microbes, with four samples returning no identifiable bacterial sequences. Our results do not provide convincing evidence for the existence of a breastmilk microbiota at six-weeks postpartum. It is more likely that microbes present in breastmilk are sourced by ejection from the infant’s mouth and from surrounding skin, as well as contamination during sampling and processing. Less
The adult spinal cord stem cell potential resides within the ependymal cell population and declines with age Ependymal cells are however heterogeneous and the biological diversity this represents and how it changes with age remain unknown Here we present a single-cell transcriptomic census of spinal cord ependymal cells from adult and aged mice identifying not only all known ependymal cell subtypes but also immature as well as mature cell states By comparing transcriptomes of spinal cord and brain ependymal cells which lack stem cell abilities we identify immature cells as potential spinal cord stem cells Following spinal cord injury these ... More
The adult spinal cord stem cell potential resides within the ependymal cell population and declines with age. Ependymal cells are, however, heterogeneous, and the biological diversity this represents and how it changes with age remain unknown. Here, we present a single-cell transcriptomic census of spinal cord ependymal cells from adult and aged mice, identifying not only all known ependymal cell subtypes but also immature as well as mature cell states. By comparing transcriptomes of spinal cord and brain ependymal cells, which lack stem cell abilities, we identify immature cells as potential spinal cord stem cells. Following spinal cord injury, these cells re-enter the cell cycle, which is accompanied by a short-lived reversal of ependymal cell maturation. We further analyze ependymal cells in the human spinal cord and identify widespread cell maturation and altered cell identities. This in-depth characterization of spinal cord ependymal cells provides insight into their biology and informs strategies for spinal cord repair. Less
Background Xylem the most abundant tissue on Earth is responsible for lateral growth in plants Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells In most angiosperms fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support while both functions are performed by tracheids in other vascular plants such as gymnosperms Little is known about the developmental programs and evolutionary relationships of these xylem cell types Results Through both single-cell and laser capture microdissection transcriptomic profiling we determine the developmental lineages of ray and fusiform cells in ... More
Background
Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types.
Results
Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers.
Conclusions
This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history. Less
Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types.
Results
Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers.
Conclusions
This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history. Less
Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity Most previous single-cell whole-genome RNA sequencing scWGS-RNA-seq methods demonstrate utility with intact cells from fresh samples Among them many are not applicable to frozen samples that cannot produce intact single-cell suspensions We have developed scONE-seq a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects We identified a unique transcriptionally normal-like tumor clone by analyzing a -year ... More
Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity. Most previous single-cell whole-genome RNA sequencing (scWGS-RNA-seq) methods demonstrate utility with intact cells from fresh samples. Among them, many are not applicable to frozen samples that cannot produce intact single-cell suspensions. We have developed scONE-seq, a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples. We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects. We identified a unique transcriptionally normal-like tumor clone by analyzing a 2-year frozen astrocytoma sample, demonstrating that performing single-cell multi-omics interrogation on biobanked tissue by scONE-seq could enable previously unidentified discoveries in tumor biology. Less
Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms SNPs The main limitation of current methods is the cost is too high to screen breeding populations Molecular inversion probes MIPs is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective high-throughput and provides high data quality to screen breeder s germplasm for genomic selection A K MIP SNP set was developed for soybean with uniformly distributed markers across the genome The SNPs were selected to maximize the number ... More
Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms (SNPs). The main limitation of current methods is the cost is too high to screen breeding populations. Molecular inversion probes (MIPs) is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective, high-throughput, and provides high data quality to screen breeder’s germplasm for genomic selection. A 1K MIP SNP set was developed for soybean with uniformly distributed markers across the genome. The SNPs were selected to maximize the number of informative markers in germplasm being tested in soybean breeding programs located in the North Central and Mid-South regions of the United States. The 1K SNP MIP set was tested on diverse germplasm and a recombinant inbred line population. Targeted sequencing with MIPs obtained an 85% enrichment for the targeted SNPs. MIP’s genotyping accuracy was 93% overall while homozoygous call accuracy was 98% with less than 10% missing data. The accuracy of MIPs combined with its low per sample cost makes it a powerful tool to enable genomic selection within soybean breeding programs. Less
Single-cell nucleosome methylome and transcriptome scNMT sequencing is a recently developed method that allows multiomics profiling of single cells In this scNMT protocol we describe profiling of cells from mouse brain and pancreatic organoids using liquid handling platforms to increase throughput from -well to -well plate format Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq protocol to obtain higher numbers of detected genes and genomic DNA gDNA CpGs per cell We outline normalization steps to optimally distribute per-cell sequencing depth
The recent discovery of comammox complete ammonia oxidation Nitrospira has upended the long-held nitrification paradigm Although comammox Nitrospira have been identified in wastewater treatment systems the conditions for their dominance over canonical ammonia oxidizers remain unclear Here we report the dominance of comammox Nitrospira in a moving bed biofilm reactor MBBR fed with synthetic mainstream wastewater Integrated S rRNA gene amplicon sequencing fluorescence in situ hybridization FISH and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen DO concentration above mg O L The dominance of comammox Nitrospira over canonical ... More
The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02–0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR. Less
Mice deficient for all ten-eleven translocation TET genes exhibit early gastrulation lethality However separating cause and effect in such embryonic failure is challenging To isolate cell-autonomous effects of TET loss we used temporal single-cell atlases from embryos with partial or complete mutant contributions Strikingly when developing within a wild-type embryo Tet-mutant cells retain near-complete differentiation potential whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential We map de-repressions of early epiblast factors e g Dppa and Gdf and failure to activate multiple signaling from nascent mesoderm Lefty FGF and Notch as likely ... More
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications. Less
The SARS-CoV- infection cycle is a multistage process that relies on functional interactions between the host and the pathogen Here we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA '-O-ribose cap needed for viral immune escape We find that the host cap '-O-ribose methyltransferase MTr can compensate for loss of viral NSP methyltransferase in facilitating virus replication Concomitant inhibition of MTr and NSP efficiently suppresses SARS-CoV- replication Using in silico target-based drug screening we identify a bispecific MTr NSP inhibitor with anti-SARS-CoV- activity in vitro and in vivo but with unfavorable ... More
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19. Less
Buffalo flies Haematobia irritans exigua are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas most commonly on the medial canthus of the eye along the lateral and ventral neck and on the abdomen of cattle For closely related horn flies Haematobia irritans irritans Staphylococcus aureus has been suggested as a contributing factor in the development of lesions To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions swabs were taken from ... More
Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. Less
Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff Twenty-six mobile phones of health care staff were swabbed DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling ... More
Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones. These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour. Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination. To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff. Twenty-six mobile phones of health care staff were swabbed. DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling was performed. Survey questionnaires were handed to the staff to collect information on mobile phone usage and users’ behaviours. Each of the 26 mobile phones of this study was contaminated with microbes with the detection of antibiotic resistance and virulent factors. Taken together the sum of microbes and genes added together across all 26 mobile phones totalised 11,163 organisms (5714 bacteria, 675 fungi, 93 protists, 228 viruses, 4453 bacteriophages) and 2096 genes coding for antibiotic resistance and virulent factors. The survey of medical staff showed that 46% (12/26) of the participants used their mobile phones in the bathroom. Mobile phones are vectors of microbes and can contribute to microbial dissemination and nosocomial diseases worldwide. As fomites, mobile phones that are not decontaminated may pose serious risks for public health and biosecurity. Less
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Understanding macrophage heterogeneity in tissue repair is a major challenge Here we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing We use a modified version of the original Smart-seq protocol to increase speed and accuracy This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states While mouse models are used extensively to investigate the interplay between microbes and the inflamed state the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships To address this issue we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria CIAMIB The initial release of this collection comprises isolates of unique bacterial species covering phyla and containing previously uncultivated isolates including novel family and novel genera The collection ... More
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states. While mouse models are used extensively to investigate the interplay between microbes and the inflamed state, the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships. To address this issue, we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria (CIAMIB). The initial release of this collection comprises 41 isolates of 39 unique bacterial species, covering 4 phyla and containing 10 previously uncultivated isolates, including 1 novel family and 7 novel genera. The collection significantly expands the number of available Muribaculaceae, Lachnospiraceae, and Coriobacteriaceae isolates and includes microbes from genera associated with inflammation, such as Prevotella and Klebsiella. We characterized the growth of CIAMIB isolates across a diverse range of nutritional conditions and predicted their metabolic potential and anaerobic fermentation capacity based on the genomes of these isolates. We also provide the first metabolic analysis of species within the genus Adlercreutzia, revealing these representatives to be nitrate-reducing and severely restricted in their ability to grow on carbohydrates. CIAMIB isolates are fully sequenced and available to the scientific community as a powerful tool to study host-microbiota interactions. Less
Many animal species are susceptible to severe acute respiratory syndrome coronavirus SARS-CoV- infection and could act as reservoirs however transmission in free-living animals has not been documented White-tailed deer the predominant cervid in North America are susceptible to SARS-CoV- infection and experimentally infected fawns can transmit the virus To test the hypothesis that SARS-CoV- is circulating in deer retropharyngeal lymph node RPLN samples collected from free-living and captive deer in Iowa from April through January of were assayed for the presence of SARS-CoV- RNA Ninety-four of the deer samples were positive for SARS-CoV- RNA as assessed by RT-PCR Notably following ... More
Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive “One Health” approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2. Less
Genetic variants of severe acute respiratory syndrome coronavirus SARS-CoV- have repeatedly altered the course of the coronavirus disease COVID- pandemic Delta variants are now the focus of intense international attention because they are causing widespread COVID- globally and are associated with vaccine breakthrough cases We sequenced SARS-CoV- genomes from samples acquired March through September in the Houston Methodist hospital system This sample represents of all Methodist system COVID- patients during the study period Delta variants increased rapidly from late April onward to cause of all COVID- cases and spread throughout the Houston metroplex Compared with all other variants combined Delta ... More
Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly altered the course of the coronavirus disease 2019 (COVID-19) pandemic. Delta variants are now the focus of intense international attention because they are causing widespread COVID-19 globally and are associated with vaccine breakthrough cases. We sequenced 16,965 SARS-CoV-2 genomes from samples acquired March 15, 2021, through September 20, 2021, in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared with all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared with 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold value (a proxy for high virus load). This value was similar to the median cycle threshold value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. The integrated analysis indicates that vaccines used in the United States are highly effective in decreasing severe COVID-19, hospitalizations, and deaths. Less
Despite advances in spatial transcriptomics the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge We present a method termed behavioral transcriptomics that allows us to couple physiological behaviors of single cells in an intact tissue to deep molecular profiling of individual cells This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following tissue injury
Squamous cell carcinoma SCC is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses AK Chronic inflammation is a well-known promoter of skin cancer progression AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus S aureus Certain secreted products from S aureus are known to promote cutaneous pro-inflammatory responses however not all S aureus strains produce these As inflammation plays a key role in SCC development we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S aureus Sterile culture supernatants secretomes of S aureus clinical ... More
Squamous cell carcinoma (SCC) is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses (AK). Chronic inflammation is a well-known promoter of skin cancer progression. AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus (S. aureus). Certain secreted products from S. aureus are known to promote cutaneous pro-inflammatory responses; however, not all S. aureus strains produce these. As inflammation plays a key role in SCC development, we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S. aureus. Sterile culture supernatants (“secretomes”) of S. aureus clinical strains isolated from AK and SCC were applied to human keratinocytes in vitro. Some S. aureus secretomes induced keratinocytes to overexpress inflammatory mediators that have been linked to skin carcinogenesis, including IL-6, IL-8, and TNFα. A large phenotypic variation between the tested clinical strains was observed. Strains that are highly pro-inflammatory in vitro also caused more pronounced skin inflammation in mice. Proteomic characterization of S. aureus secretomes using mass spectrometry established that specific S. aureus enzymes and cytolytic toxins, including hemolysins, phenol-soluble modulins, and serine proteases, as well as currently uncharacterized proteins, correlate with the pro-inflammatory S. aureus phenotype. This study is the first to describe the toxin secretion profiles of AK and SCC-associated S. aureus, and their potential to induce a pro-inflammatory environment in the skin. Further studies are needed to establish whether these S. aureus products promote SCC development by mediating chronic inflammation. Less
Certain genetic variants of severe acute respiratory syndrome coronavirus SARS-CoV- are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients SARS-CoV- genome sequences from patients in the Houston Methodist health care system diagnosed from January through May are reported here Prevalence of the B Alpha variant increased rapidly and caused to of new cases in the latter half of May Eleven B genomes had an E K replacement in spike protein a change also identified in other SARS-CoV- lineages Compared with non B -infected patients individuals with B ... More
Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non–B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. In the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex. Less
In humans epidermal melanocytes are responsible for skin pigmentation defence against ultraviolet radiation and the deadliest common skin cancer melanoma Although there is substantial overlap in melanocyte development pathways between different model organisms species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved Here we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin capturing transcriptomes across different anatomical sites developmental age sexes and multiple skin tones We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood The transcriptional signature ... More
In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy. Less
There is increasing attention focussed on the risks associated with mobile phones possibly serving as Trojan Horse fomites for microbial transmission in healthcare settings However little is reported on the presence of microbes on community derived mobile phones which in numbered in the billions in circulation with majority being used on a daily basis Identify viable microbial organisms swabbed from smartphones on a university campus Entire surfaces of mobile phones were swabbed and examined for their microbial content using pre-agar-based growths followed by downstream DNA metagenomic next-generation sequencing analysis All phones were contaminated with viable microbes bacteria fungi protists bacteriophages ... More
There is increasing attention focussed on the risks associated with mobile phones possibly serving as ‘Trojan Horse’ fomites for microbial transmission in healthcare settings. However, little is reported on the presence of microbes on community derived mobile phones which in 2021, numbered in the billions in circulation with majority being used on a daily basis. Identify viable microbial organisms swabbed from smartphones on a university campus. Entire surfaces of 5 mobile phones were swabbed and examined for their microbial content using pre-agar-based growths followed by downstream DNA metagenomic next-generation sequencing analysis. All phones were contaminated with viable microbes. 173 bacteria, 8 fungi, 8 protists, 53 bacteriophages, 317 virulence factor genes and 41 distinct antibiotic resistant genes were identified. While this research represents a pilot study, the snapshot metagenomic analysis of samples collected from the surface of mobile phones has revealed the presence of a large population of viable microbes and an array of antimicrobial resistant factors. With billions of phones in circulation, these devices might be responsible for the rise of community acquired infections. These pilot results highlight the importance of public health authorities considering mobile phones as ‘Trojan Horse’ devices for microbial transmission and ensure appropriate decontamination campaigns are implemented. Less
Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information respectively However the mechanisms underlying the assembly of such parallel networks during development remain largely unknown Our study shows that in mice complementary expression of cell surface molecules teneurin- Ten and latrophilin- Lphn in the medial and lateral hippocampal networks respectively guides the precise assembly of CA -to-subiculum connections in both networks In the medial network Ten -expressing Ten CA axons are repelled by target-derived Lphn revealing that Lphn - and Ten -mediated heterophilic repulsion and Ten -mediated homophilic attraction cooperate to control precise target selection of CA ... More
Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3→Ten3, Lphn2→Lphn2" rule instructed by reciprocal repulsions. Less
Background Psoriasis is an inflammatory IL- driven skin disease in which autoantigen-induced CD T cells have been identified as pathogenic drivers Objective Our study focused on comprehensively characterizing the phenotypic variation of CD T cells in psoriatic lesions Methods We used single-cell RNA sequencing to compare CD T-cell transcriptomic heterogeneity between psoriatic and healthy skin Results We identified transcriptionally diverse CD T-cell subsets in psoriatic and healthy skin Among several inflammatory subsets enriched in psoriatic skin we observed Tc cell subsets that were metabolically divergent were developmentally related and expressed CXCL which we found to be a biomarker of psoriasis ... More
Background Psoriasis is an inflammatory, IL-17–driven skin disease in which autoantigen-induced CD8+ T cells have been identified as pathogenic drivers. Objective Our study focused on comprehensively characterizing the phenotypic variation of CD8+ T cells in psoriatic lesions. Methods We used single-cell RNA sequencing to compare CD8+ T-cell transcriptomic heterogeneity between psoriatic and healthy skin. Results We identified 11 transcriptionally diverse CD8+ T-cell subsets in psoriatic and healthy skin. Among several inflammatory subsets enriched in psoriatic skin, we observed 2 Tc17 cell subsets that were metabolically divergent, were developmentally related, and expressed CXCL13, which we found to be a biomarker of psoriasis severity and which achieved comparable or greater accuracy than IL17A in a support vector machine classifier of psoriasis and healthy transcriptomes. Despite high coinhibitory receptor expression in the Tc17 cell clusters, a comparison of these cells with melanoma-infiltrating CD8+ T cells revealed upregulated cytokine, cytolytic, and metabolic transcriptional activity in the psoriatic cells that differed from an exhaustion program. Conclusion Using high-resolution single-cell profiling in tissue, we have uncovered the diverse landscape of CD8+ T cells in psoriatic and healthy skin, including 2 nonexhausted Tc17 cell subsets associated with disease severity. Less
The transcription factor Rora has been shown to be important for the development of ILC and the regulation of ILC macrophages and Treg cells Here we investigate the role of Rora across CD T cells in general but with an emphasis on Th cells both in vitro as well as in the context of several in vivo type infection models We dissect the function of Rora using overexpression and a CD -conditional Rora-knockout mouse as well as a RORA-reporter mouse We establish the importance of Rora in CD T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection and ... More
The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment. Less
Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle Despite their significant biogeochemical roles the microbial communities especially in the oxygen depleted compartments are poorly known Here we present a comprehensive dataset including shotgun metagenomes from stratified lakes and ponds mainly located in the boreal and subarctic regions but also including one tropical reservoir and one temperate lake For most lakes and ponds the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer The majority of the samples were collected during ... More
Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle. Despite their significant biogeochemical roles, the microbial communities, especially in the oxygen depleted compartments, are poorly known. Here, we present a comprehensive dataset including 267 shotgun metagenomes from 41 stratified lakes and ponds mainly located in the boreal and subarctic regions, but also including one tropical reservoir and one temperate lake. For most lakes and ponds, the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer. The majority of the samples were collected during the open water period, but also a total of 29 samples were collected from under the ice. In addition to the metagenomic sequences, the dataset includes environmental variables for the samples, such as oxygen, nutrient and organic carbon concentrations. The dataset is ideal for further exploring the microbial taxonomic and functional diversity in freshwater environments and potential climate change impacts on the functioning of these ecosystems. Less
Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity thousands of distinct genes with outstanding sensitivity and specificity of transcript quantification These full-length libraries have the advantage of allowing probing of transcript isoforms are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences Since full-length protocols are mostly plate-based at present they are also suited to profiling cell types where cell numbers are limiting such as rare cell types during development A disadvantage of these methods has been the scalability and cost of the experiments which has limited their ... More
Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences. Since full-length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared with droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length single-cell RNA sequencing, including both an in-house automated Smart-seq2 protocol and a commercial kit–based workflow. The protocols take 3–5 d to complete, depending on the number of plates processed in a batch. We discuss these two protocols in terms of ease of use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we have optimized the in-house automated protocol to dramatically reduce its cost. An automated setup can be adopted easily by a competent researcher with basic laboratory skills and no prior automation experience. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org). Less
Stem cell dysfunction drives many age-related disorders Identifying mechanisms that initially compromise stem cell behavior represent early targets to promote tissue function later in life Here we pinpoint multiple factors that disrupt neural stem cell NSC behavior in the adult hippocampus Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term NSCs ST-NSCs and long-term NSCs LT-NSCs ST-NSCs divide rapidly to generate neurons and deplete in the young brain Meanwhile multipotent LT-NSCs are maintained for months but are pushed out of homeostasis by lengthening quiescence Single-cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in ... More
Stem cell dysfunction drives many age-related disorders. Identifying mechanisms that initially compromise stem cell behavior represent early targets to promote tissue function later in life. Here, we pinpoint multiple factors that disrupt neural stem cell (NSC) behavior in the adult hippocampus. Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term NSCs (ST-NSCs) and long-term NSCs (LT-NSCs). ST-NSCs divide rapidly to generate neurons and deplete in the young brain. Meanwhile, multipotent LT-NSCs are maintained for months but are pushed out of homeostasis by lengthening quiescence. Single-cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in the mature brain and identified tyrosine-protein kinase Abl1 as an NSC aging factor. Treatment with the Abl inhibitor imatinib increased NSC activation without impairing NSC maintenance in the middle-aged brain. Our study indicates that hippocampal NSCs are particularly vulnerable and adaptable to cellular aging. Less
Background Huntington's disease HD is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors Using shotgun sequencing we investigated the gut microbiome composition in the R transgenic mouse model of HD from to weeks of age early adolescent through to adult stages Targeted metabolomics was also performed on the blood plasma of these mice n per group at weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile Results ... More
Background Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors. Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors. Using shotgun sequencing, we investigated the gut microbiome composition in the R6/1 transgenic mouse model of HD from 4 to 12 weeks of age (early adolescent through to adult stages). Targeted metabolomics was also performed on the blood plasma of these mice (n = 9 per group) at 12 weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile. Results Modelled time profiles of each species, KEGG Orthologs and bacterial genes, revealed heightened volatility in the R6/1 mice, indicating potential early effects of the HD mutation in the gut. In addition to gut dysbiosis in R6/1 mice at 12 weeks of age, gut microbiome function was perturbed. In particular, the butanoate metabolism pathway was elevated, suggesting increased production of the protective SCFA, butyrate, in the gut. No significant alterations were found in the plasma butyrate and propionate levels in the R6/1 mice at 12 weeks of age. The statistical integration of the metagenomics and metabolomics unraveled several Bacteroides species that were negatively correlated with ATP and pipecolic acid in the plasma. Conclusions The present study revealed the instability of the HD gut microbiome during the pre-motor symptomatic stage of the disease which may have dire consequences on the host's health. Perturbation of the HD gut microbiome function prior to significant cognitive and motor dysfunction suggest the potential role of the gut in modulating the pathogenesis of HD, potentially via specific altered plasma metabolites which mediate gut-brain signaling. Less
Single-cell RNA-sequencing technologies are ideally placed to resolve intratumoral heterogeneity However the lack of coverage across key mutation hotspots has precluded the correlation of genetic and transcriptional readouts from the same single cell To overcome this we developed TARGET-seq a protocol for TARGETed high-sensitivity single-cell mutational analysis with extremely low allelic dropout rates parallel RNA SEQuencing and cell-surface proteomics Here we present a detailed step-by-step protocol for TARGET-seq including troubleshooting tips approaches for automation and methods for high-throughput multiplexing of libraries
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types Here we examine how specific transcriptomic types of mouse prefrontal cortex PFC projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks We found that most types projected to multiple targets and most targets received projections from multiple types except PFC PAG periaqueductal gray By comparing Ca activity of the molecularly homogeneous PFC PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice ... More
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics. Less
The role of gene expression during learning and in short-term memories has been studied extensively but less is known about remote memories which can persist for a lifetime Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning Out of a vast plasticity-coding space we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory Unexpectedly astrocytes and ... More
The role of gene expression during learning and in short-term memories has been studied extensively1,2,3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage. Less
Ageing is characterised by cellular senescence leading to imbalanced tissue maintenance cell death and compromised organ function This is first observed in the thymus the primary lymphoid organ that generates and selects T cells However the molecular and cellular mechanisms underpinning these ageing processes remain unclear Here we show that mouse ageing leads to less efficient T cell selection decreased self-antigen representation and increased T cell receptor repertoire diversity Using a combination of single-cell RNA-seq and lineage-tracing we find that progenitor cells are the principal targets of ageing whereas the function of individual mature thymic epithelial cells is compromised only ... More
Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus. Less
Lung cancer the leading cause of cancer mortality exhibits heterogeneity that enables adaptability limits therapeutic success and remains incompletely understood Single-cell RNA sequencing scRNA-seq of metastatic lung cancer was performed using clinical biopsies obtained from patients before and during targeted therapy Over cancer and tumor microenvironment TME single-cell profiles exposed a rich and dynamic tumor ecosystem scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically Cancer cells surviving therapy as residual disease RD expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition whereas those present at on-therapy progressive disease PD upregulated kynurenine plasminogen and gap-junction pathways ... More
Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes. Less
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states However current RNA-seq methods are unable to simultaneously monitor both short and long poly A and poly A -transcripts at the single-cell level and thus deliver only a partial snapshot of the cellular RNAome Here we describe Smart-seq-total a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell Built upon the template-switch mechanism Smart-seq-total bears the key feature of its predecessor Smart-seq namely the ability to capture full-length transcripts ... More
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current RNA-seq methods are unable to simultaneously monitor both short and long, poly(A)+ and poly(A)-transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here, we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It also outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts alone. Less
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology these effectors are known as ROPs and GRAs respectively To examine the individual impacts of ROPs and GRAs on host gene expression we developed a robust novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected U-I cells which Toxoplasma injects with ROPs but subsequently fails to invade We then performed single-cell transcriptomic analysis at to h postinfection on U-I cells as well as on uninfected and ... More
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. Less
Single-cell RNA sequencing scRNA-seq is the leading technique for characterizing the transcriptomes of individual cells in a sample The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues organs and organisms However the protocols differ substantially with respect to their RNA capture efficiency bias scale and costs and their relative advantages for different applications are unclear In the present study we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states We performed a multicenter study comparing commonly used scRNA-seq and single-nucleus ... More
Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas. Less
Brain endothelial cells BECs are key constituents of the blood-brain barrier BBB protecting the brain from pathogens and restricting access of circulatory factors Yet because circulatory proteins have prominent age-related effects on adult neurogenesis neuroinflammation and cognitive function in mice we wondered whether BECs receive and potentially relay signals between the blood and brain Using single-cell RNA sequencing of hippocampal BECs we discover that capillary BECs compared with arterial and venous BECs undergo the greatest transcriptional changes in normal aging upregulating innate immunity and oxidative stress response pathways Short-term infusions of aged plasma into young mice recapitulate key aspects of ... More
Brain endothelial cells (BECs) are key constituents of the blood-brain barrier (BBB), protecting the brain from pathogens and restricting access of circulatory factors. Yet, because circulatory proteins have prominent age-related effects on adult neurogenesis, neuroinflammation, and cognitive function in mice, we wondered whether BECs receive and potentially relay signals between the blood and brain. Using single-cell RNA sequencing of hippocampal BECs, we discover that capillary BECs—compared with arterial and venous BECs—undergo the greatest transcriptional changes in normal aging, upregulating innate immunity and oxidative stress response pathways. Short-term infusions of aged plasma into young mice recapitulate key aspects of this aging transcriptome, and remarkably, infusions of young plasma into aged mice exert rejuvenation effects on the capillary transcriptome. Together, these findings suggest that the transcriptional age of BECs is exquisitely sensitive to age-related circulatory cues and pinpoint the BBB itself as a promising therapeutic target to treat brain disease. Less
Toxoplasma gondii a protozoan parasite undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts Here we applied single-cell RNA-sequencing scRNA-seq on Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development revealing hidden states and transcriptional factors associated with each developmental stage Analysis of SAG -related sequence SRS antigenic repertoire reveals a highly heterogeneous sporadic expression pattern unexplained by measurement noise cell cycle or asexual development Furthermore we identified AP IX- as a transcription factor that controls ... More
Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource. Less
Carbapenem-resistant Enterobacteriaceae CRE represent an urgent threat to human health Here we report the application of several complementary whole-genome sequencing WGS technologies to characterise a hospital outbreak of blaIMP- carbapenemase-producing E hormaechei Using Illumina sequencing we determined that all outbreak strains were sequence type ST and near-identical Comparison to publicly available data linked all outbreak isolates to a isolate from the same ward suggesting an environmental source in the hospital Using Pacific Biosciences sequencing we resolved the complete context of the blaIMP- gene on a large IncHI plasmid carried by all IMP- -producing strains across different hospitals Shotgun metagenomic sequencing ... More
Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak. Less
In tropical iron ore regions biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts requiring re-formation of these duricrusts However cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly A glucose-fermenting microbial consortium capable of reducing at least mmol L goethite was enriched from an iron duricrust region Metagenome analysis led to the recovery of a metagenome assembled genome MAG of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum This is the first report of ... More
In tropical iron ore regions, biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts. This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts, requiring re-formation of these duricrusts. However, cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly. A glucose-fermenting microbial consortium capable of reducing at least 27 mmol/L goethite was enriched from an iron duricrust region. Metagenome analysis led to the recovery of a metagenome assembled genome (MAG) of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum. This is the first report of iron reduction within the Telmatospirillum and the first reported genome of an iron-reducing, neutrophilic member of the Alphaproteobacteria. The Telmatospirillum MAG encodes putative metal transfer reductases (MtrA, MtrB) and a novel, multi-heme outer membrane cytochrome for extracellular electron transfer. In the presence of goethite, short chain fatty acid production shifted significantly in favor of acetate rather than propionate, indicating goethite is a hydrogen sink in the culture. Therefore, the presence of fermentative bacteria likely promotes iron reduction via hydrogen production. Stimulating microbial fermentation has potential to drive reduction of crystalline iron oxides, the rate limiting step for iron duricrust re-formation. Less
Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse with excitatory types being less layer-restricted than expected Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types Despite this general conservation we also ... More
Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain. Less
Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses To this end we have adapted the viral tagging approach which bypasses the need for culture-based methods to identify host phage pairings Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells which are then individually sorted as host phage pairs followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus es We demonstrate single-cell viral tagging ... More
Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses. One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses. To this end, we have adapted the viral tagging approach, which bypasses the need for culture-based methods to identify host–phage pairings. Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells, which are then individually sorted as host–phage pairs, followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus(es). We demonstrate single-cell viral tagging using the faecal microbiome, including cross-tagging of viruses and bacteria between human subjects. A total of 363 unique host–phage pairings were predicted, most of which were subject-specific and involved previously uncharacterized viruses despite the majority of their bacterial hosts having known taxonomy. One-fifth of these pairs were confirmed by multiple individual tagged cells. Viruses targeting more than one bacterial species were conspicuously absent in the host–phage network, suggesting that phages are not major vectors of inter-species horizontal gene transfer in the human gut. A high level of cross-reactivity between phages and bacteria from different subjects was noted despite subject-specific viral profiles, which has implications for faecal microbiota transplant therapy. Less
RNase H dependent PCR-enabled T-cell receptor sequencing rhTCRseq can be used to determine paired alpha beta T-cell receptor TCR clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples With the enhanced specificity of RNase H dependent PCR rhPCR it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step For single cells the protocol includes sorting of single cells into plates generation of cDNA libraries a TCR-specific amplification step a second PCR on pooled sample to generate a sequencing library and sequencing In the bulk method sorting and cDNA library ... More
RNase H–dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H–dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2–4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d. Less
An aged circulatory environment can activate microglia reduce neural precursor cell activity and impair cognition in mice We hypothesized that brain endothelial cells BECs mediate at least some of these effects We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule VCAM a protein that facilitates vascular immune cell interactions Concomitantly levels of the shed soluble form of VCAM are prominently increased in the plasma of aged humans and mice and their plasma is sufficient to increase VCAM expression in cultured BECs and the hippocampi of young mice ... More
An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular–immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood–brain barrier as a possible target to treat age-related neurodegeneration. Less
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions Here we performed deep single-cell RNA sequencing scRNA-seq of microglia and related myeloid cells sorted from various regions of embryonic early postnatal and adult mouse brains We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes regardless of brain region By contrast early postnatal microglia are more heterogeneous We discovered a proliferative-region-associated microglia PAM ... More
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease. Less
High-throughput single-cell RNA-seq methods assign limited unique molecular identifier UMI counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts We thus developed a high-throughput single-cell RNA-seq method Quartz-Seq to overcome these issues Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts at a rate of and detect more genes To demonstrate the power of Quartz-Seq we analyzed approximately transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads
Methods are provided for analyzing one or more genetic samples comprising procuring one or more genetic samples comprising genetic material from one or more individuals and sequencing the genetic material using non-targeted ultra-low coverage sequencing to obtain genetic information for individual associated with the one or more genetic samples Personal and genetic information associated with the individuals is stored in a database for retrieval and manipulation
In recent years highly detailed characterization of adult bone marrow BM myeloid progenitors has been achieved and as a result the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined Fetal liver FL hematopoietic progenitor cells HPCs are poorly characterized in comparison potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis Numerous disorders for example infant acute leukemias have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets We previously demonstrated that a Runx distal promoter P -GFP proximal promoter P -hCD ... More
In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible. Less