Direct imaging of the circular chromosome in a live bacterium, bioRxiv, 2018-01-12
New assays for quantitative imaging1–6 and sequencing7–11 have yielded great progress towards understanding the organizational principles of chromosomes. Yet, even for the well-studied model bacterium Escherichia coli, many basic questions remain unresolved regarding chromosomal (sub-)structure2,11, its mechanics1,2,12 and dynamics13,14, and the link between structure and function1,15,16. Here we resolve the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to exhibit a torus topology with a 4.2 μm toroidal length and 0.4 μm bundle thickness. On average, the DNA density along the chromosome shows dense right and left arms that branch from a lower-density origin of replication, and are connected at the terminus of replication by an ultrathin flexible string of DNA. At the single-cell level, the DNA density along the torus is found to be strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. We show that prominent domain boundaries at the terminus and origin of replication are induced by MatP proteins, while weaker transient domain boundaries are facilitated by the global transcription regulators HU and Fis. These findings provide an architectural basis for the understanding of the spatial organization of bacterial genomes.
biorxiv microbiology 100-200-users 2018High accuracy haplotype-derived allele frequencies from ultra-low coverage pool-seq samples, bioRxiv, 2018-01-12
AbstractEvolve-and-resequence experiments leverage next-generation sequencing technology to track allele frequency dynamics of populations as they evolve. While previous work has shown that adaptive alleles can be detected by comparing frequency trajectories from many replicate populations, this power comes at the expense of high-coverage (>100x) sequencing of many pooled samples, which can be cost-prohibitive. Here we show that accurate estimates of allele frequencies can be achieved with very shallow sequencing depths (<5x) via inference of known founder haplotypes in small genomic windows. This technique can be used to efficiently estimate frequencies for any number of alleles in any model system. Using both experimentally-pooled and simulated samples of Drosophila melanogaster, we show that haplotype inference can improve allele frequency accuracy by orders of magnitude, and that high accuracy is maintained after up to 200 generations of recombination, even in the presence of missing data or incomplete founder knowledge. By reducing sequencing costs without sacrificing accuracy, our method enables analysis of samples from more time-points and replicates, increasing the statistical power to detect adaptive alleles.
biorxiv evolutionary-biology 0-100-users 2018Limitation of alignment-free tools in total RNA-seq quantification, bioRxiv, 2018-01-12
AbstractBackgroundAlignment-free RNA quantification tools have significantly increased the speed of RNA-seq analysis. However, it is unclear whether these state-of-the-art RNA-seq analysis pipelines can quantify small RNAs as accurately as they do with long RNAs in the context of total RNA quantification.ResultWe comprehensively tested and compared four RNA-seq pipelines on the accuracies of gene quantification and fold-change estimation on a novel total RNA benchmarking dataset, in which small non-coding RNAs are highly represented along with other long RNAs. The four RNA-seq pipelines were of two commonly-used alignment-free pipelines and two variants of alignment-based pipelines. We found that all pipelines showed high accuracies for quantifying the expressions of long and highly-abundant genes. However, alignment-free pipelines showed systematically poorer performances in quantifying lowly-abundant and small RNAs.ConclusionWe have shown that alignment-free and traditional alignment-based quantification methods performed similarly for common gene targets, such as protein-coding genes. However, we identified a potential pitfall in analyzing and quantifying lowly-expressed genes and small RNAs with alignment-free pipelines, especially when these small RNAs contain mutations.
biorxiv bioinformatics 100-200-users 2018Widespread and targeted gene expression by systemic AAV vectors Production, purification, and administration, bioRxiv, 2018-01-12
ABSTRACTWe recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label andor genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.
biorxiv neuroscience 0-100-users 2018Easy Hi-C A simple efficient protocol for 3D genome mapping in small cell populations, bioRxiv, 2018-01-11
Despite the growing interest in studying the mammalian genome organization, it is still challenging to map the DNA contacts genome-wide. Here we present easy Hi-C (eHi-C), a highly efficient method for unbiased mapping of 3D genome architecture. The eHi-C protocol only involves a series of enzymatic reactions and maximizes the recovery of DNA products from proximity ligation. We show that eHi-C can be performed with 0.1 million cells and yields high quality libraries comparable to Hi-C.
biorxiv genomics 0-100-users 2018A metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities, bioRxiv, 2018-01-10
AbstractMeasurements of the carbon stable isotope ratio (δ13C) are widely used in biology to address major questions regarding food sources and metabolic pathways used by organisms. Measurement of these so called stable carbon isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals have led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide limited taxonomic resolution, such as with the use of lipid biomarkers, or are limited in throughput, such as NanoSIMS imaging of single cells.Here we present “direct Protein-SIF” and the Calis-p software package (<jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpssourceforge.netprojectscalis-p>httpssourceforge.netprojectscalis-p<jatsext-link>), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we show that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis showed that direct Protein-SIF confirms previous physiological hypotheses and can provide unexpected new insights into the symbionts’ metabolism. This confirms the usefulness of this new approach to accurately determine δ13C values for different species in microbial community samples.<jatssec id=s1>SignificanceTo understand the roles that microorganisms play in diverse environments such as the open ocean and the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct Protein-SIF method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct Protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2.
biorxiv microbiology 0-100-users 2018