Muhammad S. Shamim

Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments

Hi-C experiments explore the 3D structure of the genome, generating terabases of data to create high-resolution contact maps. Here, we introduce Juicer, an open-source tool for analyzing terabase-scale Hi-C datasets. Juicer allows users without a computational background to transform raw sequence data into normalized contact maps with one click. Juicer produces a hic file containing compressed contact matrices at many resolutions, facilitating visualization and analysis at multiple scales. Structural features, such as loops and domains, are automatically annotated. Juicer is available as open source software at http://aidenlab.org/juicer/.

Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture.

Significance In human females, one of the two X chromosomes is inactive (Xi) and adopts an unusual 3D conformation. The Xi chromosome contains superloops, large chromatin loops that are often anchored at the macrosatellite repeat DXZ4, and is partitioned into two large intervals, called superdomains, whose boundary lies at DXZ4. Here, we use spatial proximity mapping, microscopy, and genome editing to study the Xi. We find that superloops and superdomains are conserved across humans, macaque, and mouse. By mapping proximity between three or more loci, we show that superloops tend to occur simultaneously. Deletion of DXZ4 from the human Xi disrupts superloops, eliminates superdomains, and alters chromatin modifications. Finally, we show that a model in which CCCTC-binding factor (CTCF) and cohesin extrude chromatin can explain the formation of superloops and superdomains. During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the “Barr body.” Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called “superdomains,” such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called “superloops.” DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds

Hi-C for mosquito genomes Most genomes sequenced today are determined through the generation of short sequenced bits of DNA that are computationally pieced together like a jigsaw puzzle. This has resulted in the need for funds and additional data to fill in gaps in order to fully assemble the many chromosomes that make up a eukaryotic genome. Dudchenko et al. used the Hi-C method, which measures the distance between contact points within and between chromosomes for scaffold validation, together with correction and ordering to more completely determine the arrangement of short sequencing reads for genome mapping. They validated their approach through the de novo generation of a complete human genome. A comparative analysis of mosquito genomes was made possible by improving the Culex quinquefasciatus genome assembly and generating the genome of Aedes aegypti, the vector of Zika virus. Science, this issue p. 92 The DNA proximity ligation method Hi-C was used to create a genome assembly for the mosquito Aedes aegypti. The Zika outbreak, spread by the Aedes aegypti mosquito, highlights the need to create high-quality assemblies of large genomes in a rapid and cost-effective way. Here we combine Hi-C data with existing draft assemblies to generate chromosome-length scaffolds. We validate this method by assembling a human genome, de novo, from short reads alone (67× coverage). We then combine our method with draft sequences to create genome assemblies of the mosquito disease vectors Ae. aegypti and Culex quinquefasciatus, each consisting of three scaffolds corresponding to the three chromosomes in each species. These assemblies indicate that almost all genomic rearrangements among these species occur within, rather than between, chromosome arms. The genome assembly procedure we describe is fast, inexpensive, and accurate, and can be applied to many species.We present an end-to-end genome assembly of a female Aedes aegypti mosquito, which spreads viral diseases such as yellow fever, dengue, chikungunya, and Zika to humans. The assembly is based on an earlier genome published in 2007 and improved in 2013. The new assembly has a scaffold N50 of 419Mb, with 96.9% of the ungapped sequence anchored to chromosomes. We used the new assembly to examine the conservation of A. aegypti chromosomes. Our results suggest that synteny is strongly conserved between Ae. aegypti and An. gambiae. Comparison to D. melanogaster highlights the extent to which the identity of entire chromosome arms is preserved across dipterans. Main text: Due to its role in the spread of the Zika virus in the Americas, A. aegypti – an important mosquito vector of many human diseases – is causing a new wave of widespread concern (Harmon 2016). The lack of an end-to-end genome assembly is limiting our understanding of the biology of this major arbovirus vector and hinders efforts at disease control. To aid in the response, we present an improved assembly. Our assembly is based on AaegL2 (Nene et al. 2007), which was generated using Sanger reads (8X coverage) assembled using ARACHNE (Jaffe et al. 2003). The AaegL2 assembly consists of 4756 scaffolds spanning 1.3Gb of sequence, with a contig N50 of 82 Kb and scaffold N50 of 1.5Mb. Our effort to improve AaegL2 resulted in the first end-to-end genome assembly of A. aegypti. We refer to our new assembly as AaegL4. (AaegL3 is frequently used to refer to a variant of AaegL2 which includes the mitochondrial genome.) In addition to providing an end-to-end assembly, AaegL4 also improves the quality of anchoring and scaffolding. The AaegL4 assembly is shared at https://tinyurl.com/AaegL4. Given the public health relevance of this work, we choose to share this assembly with the scientific community without delay. A description of the methodology used to generate AaegL4 is currently in preparation.

Cohesin Loss Eliminates All Loop Domains, Leading To Links Among Superenhancers And Downregulation Of Nearby Genes

The human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two loci - almost always bound by CTCF and cohesin – lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loci in different compartments that had been in the same loop domain become more segregated. Loss of loop domains does not lead to widespread ectopic gene activation, but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes, and affecting the regulation of nearby genes. Cohesin restoration quickly reverses these effects, consistent with a model where loop extrusion is rapid.

Hybrid de novo genome assembly and centromere characterization of the gray mouse lemur (Microcebus murinus)

BackgroundThe de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly.MethodsWe used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome.ResultsWe improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies.ConclusionsWe describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes.

A Cell Type-Specific Class of Chromatin Loops Anchored at Large DNA Methylation Nadirs

Higher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we found that large (~10kb) DNA methylation nadirs can form long loops connecting anchor loci that may be dozens of megabases apart, as well as interchromosomal links. The interacting loci comprise ~3.5Mb of the human genome. The data are more consistent with the formation of these loops by phase separation of the interacting loci to form a genomic subcompartment, rather than with CTCF-mediated extrusion. Interestingly, unlike previously characterized genomic subcompartments, this subcompartment is only present in particular cell types, such as stem and progenitor cells. Further, we identify one particular loop anchor that is functionally associated with maintenance of the hematopoietic stem cell state. Our work reveals that H3K27me3-marked large DNA methylation nadirs represent a novel set of very long-range loops and links associated with cellular identity. Summary Hi-C and DNA methylation analyses reveal novel chromatin loops between distant sites implicated in stem and progenitor cell function.

EndoC-βH1 multi-genomic profiling defines gene regulatory programs governing human pancreatic β cell identity and function

EndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g., PDX1, ISL1) and putative (e.g., PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptional cis-regulatory networks, and identify allelic effects on cis-regulatory element use and expression. Importantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures in EndoC-βH1. Together, these maps, and an interactive web application we have created for their exploration, provide important tools for the broad community in the design and success of experiments to probe and manipulate the genetic programs governing beta cell identity and (dys)function in diabetes.

A comprehensive mathematical framework for modeling intestinal smooth muscle cell contraction with applications to intestinal edema.

The contraction of intestinal smooth muscle cells (ISMCs) involves many coordinated biochemical and mechanical processes. In this work, we present a framework for modeling ISMC contractility that begins with chemical models of calcium dynamics, continues with myosin light chain phosphorylation and force generation, and ends with a cell model of the ISMC undergoing contraction-relaxation. The motivation for developing this comprehensive framework is to study the effects of edema (excess fluid build-up in the muscle tissue) on ISMC contractility. The hypothesis is that more fluid equates to dilution of an external stimulis, eventually leading to reduced contractility. We compare our results to experimental data collected from normal versus edematous intestinal muscle tissue.

The Juicebox Assembly Tools module facilitates de novo assembly of mammalian genomes with chromosome-length scaffolds for under

Hi-C contact maps are valuable for genome assembly (Lieberman-Aiden, van Berkum et al. 2009; Burton et al. 2013; Dudchenko et al. 2017). Recently, we developed Juicebox, a system for the visual exploration of Hi-C data (Durand, Robinson et al. 2016), and 3D-DNA, an automated pipeline for using Hi-C data to assemble genomes (Dudchenko et al. 2017). Here, we introduce “Assembly Tools,” a new module for Juicebox, which provides a point-and-click interface for using Hi-C heatmaps to identify and correct errors in a genome assembly. Together, 3D-DNA and the Juicebox Assembly Tools greatly reduce the cost of accurately assembling complex eukaryotic genomes. To illustrate, we generated de novo assemblies with chromosome-length scaffolds for three mammals: the wombat, Vombatus ursinus (3.3Gb), the Virginia opossum, Didelphis virginiana (3.3Gb), and the raccoon, Procyon lotor (2.5Gb). The only inputs for each assembly were Illumina reads from a short insert DNA-Seq library (300 million Illumina reads, maximum length 2x150 bases) and an in situ Hi-C library (100 million Illumina reads, maximum read length 2x150 bases), which cost <

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