Biology subjects

Ecker, J. R.

Publications and source records attributed to Ecker, J. R..

6 recordsLinked to original sources

bioRxiv2018

Multi-omic profiling of transcriptome and DNA methylome in single nuclei with molecular partitioning

Single-cell transcriptomic and epigenomic analyses provide powerful strategies for unbiased determination of cell types in mammalian tissues. Although previous studies have identified cell types using individual molecular signatures, the generation of consensus cell type classification requires the integration of multiple data types. Most existing single-cell techniques can only make one type of molecular measurement. Here we describe single-nucleus methylcytosine and transcriptome sequencing (snmCT-seq), a multi-omic method that requires no physical separation of DNA and RNA molecules. We demonstrated that snmCT-seq profiles generated from single cells or nuclei robustly distinguish human cell types and accurately measures cytosine DNA methylation and gene expression signatures of each cell type.

genomics↗

bioRxiv2018

Global DNA methylation remodeling during direct reprogramming of fibroblasts to neurons

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. In this study, we characterized global epigenomic changes during direct reprogramming using whole-genome base-resolution DNA methylome (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced strong promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

neuroscience↗

bioRxiv2018

Robust single-cell DNA methylome profiling with snmC-seq2

Single-cell DNA methylome profiling has enabled the study of epigenomic heterogeneity in complex tissues and during cellular reprogramming. However, broader applications of the method have been impeded by the modest quality of sequencing libraries. Here we report snmC-seq2, which provides improved read mapping, reduced artifactual reads, enhanced throughput, as well as increased library complexity and coverage uniformity compared to snmC-seq. snmC-seq2 is an efficient strategy suited for large scale single-cell epigenomic studies.

genomics↗

bioRxiv2018

The complex architecture of plant transgene insertions

Over the last 35 years the soil bacterium Agrobacterium tumefaciens has been the workhorse tool for plant genome engineering. Replacement of native tumor-inducing (Ti) plasmid elements with customizable cassettes enabled insertion of a sequence of interest called Transfer DNA (T-DNA) into any plant genome. Although these T-DNA transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. To fill this gap, we analyzed transgenic Arabidopsis thaliana lines from three widely used collections (SALK, SAIL and WISC) with two single molecule technologies, optical genome mapping and nanopore sequencing. Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and for the first time the actual length of individual transgene insertions from 27 to 236 kilobases. De novo nanopore sequencing-based genome assemblies for two segregating lines resolved T-DNA structures up to 36 kb into the insertions and revealed large-scale T-DNA associated translocations and exchange of chromosome arm ends. The multiple internally rearranged nature of T-DNA arrays made full assembly impossible, even with long nanopore reads. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. This unprecedented nucleotide-level definition of T-DNA insertions enabled the mapping of epigenome data. We identify variable small RNA transgene targeting and DNA methylation. SALK_059379 T-DNA insertions were enriched for 24nt siRNAs and contained dense cytosine DNA methylation. Transgene silencing via the RNA-directed DNA methylation pathway was confirmed by in planta assays. In contrast, SAIL_232 T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. With the emergence of genome editing technologies that rely on Agrobacterium for gene delivery, this study provides new insights into the structural impact of engineering plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

plant biology↗

bioRxiv2017

Systematic mapping of chromatin state landscapes during mouse development

Embryogenesis requires epigenetic information that allows each cell to respond appropriately to developmental cues. Histone modifications are core components of a cells epigenome, giving rise to chromatin states that modulate genome function. Here, we systematically profile histone modifications in a diverse panel of mouse tissues at 8 developmental stages from 10.5 days post conception until birth, performing a total of 1,128 ChIP-seq assays across 72 distinct tissue-stages. We combine these histone modification profiles into a unified set of chromatin state annotations, and track their activity across developmental time and space. Through integrative analysis we identify dynamic enhancers, reveal key transcriptional regulators, and characterize the role of chromatin-based repression in developmental gene regulation. We also leverage these data to link enhancers to putative target genes, revealing connections between coding and non-coding sequence variation in disease etiology. Our study provides a compendium of resources for biomedical researchers, and achieves the most comprehensive view of embryonic chromatin states to date.

genomics↗

bioRxiv2017

High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell

While many evolutionary questions can be answered by short read re-sequencing, presence/absence polymorphisms of genes and/or transposons have been largely ignored in large-scale intraspecific evolutionary studies. To enable the rigorous analysis of such variants, multiple high quality and contiguous genome assemblies are essential. Similarly, while genome assemblies based on short reads have made genomics accessible for non-reference species, these assemblies have limitations due to low contiguity. Long-read sequencers and long-read technologies have ushered in a new era of genome sequencing where the lengths of reads exceed those of most repeats. However, because these technologies are not only costly, but also time and compute intensive, it has been unclear how scalable they are. Here we demonstrate a fast and cost effective reference assembly for an Arabidopsis thaliana accession using the USB-sized Oxford Nanopore MinION sequencer and typical consumer computing hardware (4 Cores, 16Gb RAM). We assemble the accession KBS-Mac-74 into 62 contigs with an N50 length of 12.3 Mb covering 100% (119 Mb) of the non-repetitive genome. We demonstrate that the polished KBS-Mac-74 assembly is highly contiguous with BioNano optical genome maps, and of high per-base quality against a likewise polished Pacific Biosciences long-read assembly. The approach we implemented took a total of four days at a cost of less than 1,000 USD for sequencing consumables including instrument depreciation.

genomics↗