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Abstract Phased genomes and pangenomes are enhancing our understanding of genetic variation. Accurate phasing and assembly in repetitive regions of the genome remain challenging, however. Addressing this obstacle is crucial for studying structural genomic variation, such as copy number variations (CNVs) common to repetitive regions. Polar fishes, for example, evolved repetitive tandem arrays of antifreeze protein (AFP) genes that facilitate adaptation to freezing and expanded in copy number in colder environments. AFP CNVs remain poorly characterized in polar fishes and may be illuminated by haplotype-aware approaches. We performed long-read sequencing for two polar fishes in the suborder Zoarcoidei and leveraged additional published long-read data to assemble phased genomes. We developed a workflow to measure haplotype diversity in CNV while controlling for misassembly and switch errors—a change from one parental haplotype to another in a contiguous assembly. We presentgfa_parser, which computes and extracts all possible contiguous sequences for phased or primary assemblies from graphical fragment assembly (GFA) files, andswitch_error_screen, which flags potential switch errors.gfa_parserrevealed that assembly uncertainty was ubiquitous across AFP array haplotypes and that standard processing of graphical fragment assemblies can bias measurement of haplotype CNVs. We detected no switch errors in AFP arrays. After controlling for misassembly and switch error, we detected haplotype diversity of AFP CNVs in all studied polar Zoarcoidei species and in 60% of AFP arrays. Intraindividual haplotype diversity spanned differences of 3–16 copies. Our workflow revealed intraspecific genomic diversity in zoarcoids that likely fueled the evolution of AFP copy number across temperature.
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CiFi: accurate long-read chromosome conformation capture with low-input requirements
Abstract Hi-C characterizes three-dimensional chromatin organization, facilitates haplotype phasing, and enables genome-assembly scaffolding, but encounters difficulties across complex regions. By coupling chromosome conformation capture (3C) with PacBio HiFilong-read sequencing, here we develop a method (CiFi) that enables analysis of genomic interactions across repetitive regions. Starting with as little as 60,000 cells (sub-microgram DNA), the method produces multi-kilobasepair HiFi reads that contain multiple interacting, concatenated segments (~350 bp to 2 kbp). This multiplicity and increase in segment length versus standard short-read-based Hi-C improves read-mapping efficiency and coverage in repetitive regions and enhances haplotype phasing. CiFi pairwise interactions are largely concordant with Hi-C from a human lymphoblastoid cell line, with gains in assigning topologically associating domains across centromeres, segmental duplications, and human disease-associated genomic hotspots. As CiFi requires less input versus established methods, we apply the approach to characterize single small insects: assaying chromatin interactions across the genome from anAnopheles coluzziimosquito and producing a chromosome-scale scaffolded assembly from aCeratitis capitataMediterranean fruit fly. Together, CiFi enables assessment of chromosome-scale interactions of previously recalcitrant low-complexity loci, low-input samples, and small organisms.
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- Award ID(s):
- 2145885
- PAR ID:
- 10657972
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 17
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Zhang, Zhihua (Ed.)Recent advances in high-throughput chromosome conformation capture (Hi-C) techniques have allowed us to map genome-wide chromatin interactions and uncover higher-order chromatin structures, thereby shedding light on the principles of genome architecture and functions. However, statistical methods for detecting changes in large-scale chromatin organization such as topologically associating domains (TADs) are still lacking. Here, we proposed a new statistical method, DiffGR, for detecting differentially interacting genomic regions at the TAD level between Hi-C contact maps. We utilized the stratum-adjusted correlation coefficient to measure similarity of local TAD regions. We then developed a nonparametric approach to identify statistically significant changes of genomic interacting regions. Through simulation studies, we demonstrated that DiffGR can robustly and effectively discover differential genomic regions under various conditions. Furthermore, we successfully revealed cell type-specific changes in genomic interacting regions in both human and mouse Hi-C datasets, and illustrated that DiffGR yielded consistent and advantageous results compared with state-of-the-art differential TAD detection methods. The DiffGR R package is published under the GNU General Public License (GPL) ≥ 2 license and is publicly available at https://github.com/wmalab/DiffGR.more » « less
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null (Ed.)Abstract Background The Aedes aegypti mosquito is a threat to human health across the globe. The A. aegypti genome was recently re-sequenced and re-assembled. Due to a combination of long-read PacBio and Hi-C sequencing, the AaegL5 assembly is chromosome complete and significantly improves the assembly in key areas such as the M/m sex-determining locus. Release of the updated genome assembly has precipitated the need to reprocess historical functional genomic data sets, including cis -regulatory element (CRE) maps that had previously been generated for A. aegypti. Results We re-processed and re-analyzed the A. aegypti whole embryo FAIRE seq data to create an updated embryonic CRE map for the AaegL5 genome. We validated that the new CRE map recapitulates key features of the original AaegL3 CRE map. Further, we built on the improved assembly in the M/m locus to analyze overlaps of open chromatin regions with genes. To support the validation, we created a new method (PeakMatcher) for matching peaks from the same experimental data set across genome assemblies. Conclusion Use of PeakMatcher software, which is available publicly under an open-source license, facilitated the release of an updated and validated CRE map, which is available through the NIH GEO. These findings demonstrate that PeakMatcher software will be a useful resource for validation and transferring of previous annotations to updated genome assemblies.more » « less
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INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implemented a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. 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Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. 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