Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent–child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average contig N50: 26 Mbp) integrate all forms of genetic variation even across complex loci. We identify 107,590 structural variants (SVs), of which 68% are not discovered by short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterize 130 of the most active mobile element source elements and find that 63% of all SVs arise by homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1,526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.
A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...
Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the expression of sexspecific lincRNAs and their potential for regulation of sex differences in liver physiology and disease. High-throughput sequencing of mammalian transcriptomes has revealed nearly ubiquitous transcription of the genome and the generation of large numbers of noncoding transcripts. Noncoding RNAs (ncRNAs) have drawn much attention as potential chromatin regulators, exemplified by classical ncRNAs, such as Xist (1). Several thousand ncRNAs have been discovered in human (2, 3), mouse (4-7), zebrafish (8,9), and fruit fly (10,11). Individual ncRNAs were shown to play diverse regulatory roles in gene expression (9,(12)(13)(14)(15); however, the vast majority of ncRNAs are poorly characterized, both computationally and experimentally. Many ncRNAs share salient features of protein-coding genes, including transcription by RNA polymerase II, 5= capping, splicing, polyadenylation, and deposition of histone marks associated with transcription, specifically H3K4me3 at the promoter and H3K36me3 across the gene body (4). These ncRNAs are typically Ͼ200 nucleo...
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