Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2, bioRxiv, 2020-01-12

AbstractSingle cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5’ or 3’ end. These cDNA pools are currently analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but all current implementations rely heavily on Illumina short-reads for the analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 9 million full-length cDNA molecules generated by the 10X Chromium platform from ∼3000 peripheral blood mononuclear cells (PBMCs). We used these reads to – independent from Illumina data – cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell-types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptor – T cell receptor and B cell receptor (TCR and BCR) –sequences unique to each T and B cell. This work represents a new, simple, and powerful approach that –using a single sequencing method – can extract an unprecedented amount of information from thousands of single cells.

biorxiv genomics 100-200-users 2020

Dissecting the collateral damage of antibiotics on gut microbes, bioRxiv, 2020-01-10

AbstractAntibiotics are used for fighting pathogens, but also target our commensal bacteria as a side effect, disturbing the gut microbiota composition and causing dysbiosis and disease1-3. Despite this well-known collateral damage, the activity spectrum of the different antibiotic classes on gut bacteria remains poorly characterized. Having monitored the activities of >1,000 marketed drugs on 38 representative species of the healthy human gut microbiome4, we here characterize further the 144 antibiotics therein, representing all major classes. We determined >800 Minimal Inhibitory Concentrations (MICs) and extended the antibiotic profiling to 10 additional species to validate these results and link to available data on antibiotic breakpoints for gut microbes. Antibiotic classes exhibited distinct inhibition spectra, including generation-dependent effects by quinolones and phylogeny-independence by β-lactams. Macrolides and tetracyclines, two prototypic classes of bacteriostatic protein synthesis inhibitors, inhibited almost all commensals tested. We established that both kill different subsets of prevalent commensal bacteria, and cause cell lysis in specific cases. This species-specific activity challenges the long-standing divide of antibiotics into bactericidal and bacteriostatic, and provides a possible explanation for the strong impact of macrolides on the gut microbiota composition in animals5-8 and humans9-11. To mitigate the collateral damage of macrolides and tetracyclines on gut commensals, we exploited the fact that drug combinations have species-specific outcomes in bacteria12 and sought marketed drugs, which could antagonize the activity of these antibiotics in abundant gut commensal species. By screening >1,000 drugs, we identified several such antidotes capable of protecting gut species from these antibiotics without compromising their activity against relevant pathogens. Altogether, this study broadens our understanding of antibiotic action on gut commensals, uncovers a previously unappreciated and broad bactericidal effect of prototypical bacteriostatic antibiotics on gut bacteria, and opens avenues for preventing the collateral damage caused by antibiotics on human gut commensals.

biorxiv microbiology 100-200-users 2020

Glacier ice archives fifteen-thousand-year-old viruses, bioRxiv, 2020-01-07

AbstractWhile glacier ice cores provide climate information over tens to hundreds of thousands of years, study of microbes is challenged by ultra-low-biomass conditions, and virtually nothing is known about co-occurring viruses. Here we establish ultra-clean microbial and viral sampling procedures and apply them to two ice cores from the Guliya ice cap (northwestern Tibetan Plateau, China) to study these archived communities. This method reduced intentionally contaminating bacterial, viral, and free DNA to background levels in artificial-ice-core control experiments, and was then applied to two authentic ice cores to profile their microbes and viruses. The microbes differed significantly across the two ice cores, presumably representing the very different climate conditions at the time of deposition that is similar to findings in other cores. Separately, viral particle enrichment and ultra-low-input quantitative viral metagenomic sequencing from ∼520 and ∼15,000 years old ice revealed 33 viral populations (i.e., species-level designations) that represented four known genera and likely 28 novel viral genera (assessed by gene-sharing networks). In silico host predictions linked 18 of the 33 viral populations to co-occurring abundant bacteria, including Methylobacterium, Sphingomonas, and Janthinobacterium, indicating that viruses infected several abundant microbial groups. Depth-specific viral communities were observed, presumably reflecting differences in the environmental conditions among the ice samples at the time of deposition. Together, these experiments establish a clean procedure for studying microbial and viral communities in low-biomass glacier ice and provide baseline information for glacier viruses, some of which appear to be associated with the dominant microbes in these ecosystems.ImportanceThis study establishes ultra-clean microbial and viral sampling procedures for glacier ice, which complements prior in silico decontamination methods and expands, for the first time, the clean procedures to viruses. Application of these methods to glacier ice confirmed prior common microbiological findings for a new ice core climate record, and provides a first window into viral genomes and their ecology from glacier ice across two time horizons, and emphasizes their likely impact on abundant microbial groups. Together these efforts provide clean sampling approaches and foundational datasets that should enable simultaneous access to an archived virosphere in glacier ice.

biorxiv ecology 100-200-users 2020

 

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