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. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. 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. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx.
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Multiple genome alignment in the telomere-to-telomere assembly era
Abstract With the arrival of telomere-to-telomere (T2T) assemblies of the human genome comes the computational challenge of efficiently and accurately constructing multiple genome alignments at an unprecedented scale. By identifying nucleotides across genomes which share a common ancestor, multiple genome alignments commonly serve as the bedrock for comparative genomics studies. In this review, we provide an overview of the algorithmic template that most multiple genome alignment methods follow. We also discuss prospective areas of improvement of multiple genome alignment for keeping up with continuously arriving high-quality T2T assembled genomes and for unlocking clinically-relevant insights.
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- Award ID(s):
- 2126387
- NSF-PAR ID:
- 10378493
- Date Published:
- Journal Name:
- Genome Biology
- Volume:
- 23
- Issue:
- 1
- ISSN:
- 1474-760X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The spatiotemporal organization of DNA replication produces a highly robust and reproducible replication timing profile. Sequencing-based methods for assaying replication timing genome-wide have become commonplace, but regions of high repeat content in the human genome have remained refractory to analysis. Here, we report the first nearly-gapless telomere-to-telomere replication timing profiles in human, using the T2T-CHM13 genome assembly and sequencing data for five cell lines. We find that replication timing can be successfully assayed in centromeres and large blocks of heterochromatin. Centromeric regions replicate in mid-to-late S-phase and contain replication-timing peaks at a similar density to other genomic regions, while distinct families of heterochromatic satellite DNA differ in their bias for replicating in late S-phase. The high degree of consistency in centromeric replication timing across chromosomes within each cell line prompts further investigation into the mechanisms dictating that some cell lines replicate their centromeres earlier than others, and what the consequences of this variation are.
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INTRODUCTION One of the central applications of the human reference genome has been to serve as a baseline for comparison in nearly all human genomic studies. Unfortunately, many difficult regions of the reference genome have remained unresolved for decades and are affected by collapsed duplications, missing sequences, and other issues. Relative to the current human reference genome, GRCh38, the Telomere-to-Telomere CHM13 (T2T-CHM13) genome closes all remaining gaps, adds nearly 200 million base pairs (Mbp) of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome for scientific inquiry. RATIONALE We demonstrate how the T2T-CHM13 reference genome universally improves read mapping and variant identification in a globally diverse cohort. This cohort includes all 3202 samples from the expanded 1000 Genomes Project (1KGP), sequenced with short reads, as well as 17 globally diverse samples sequenced with long reads. By applying state-of-the-art methods for calling single-nucleotide variants (SNVs) and structural variants (SVs), we document the strengths and limitations of T2T-CHM13 relative to its predecessors and highlight its promise for revealing new biological insights within technically challenging regions of the genome. RESULTS Across the 1KGP samples, we found more than 1 million additional high-quality variants genome-wide using T2T-CHM13 than with GRCh38. Within previously unresolved regions of the genome, we identified hundreds of thousands of variants per sample—a promising opportunity for evolutionary and biomedical discovery. T2T-CHM13 improves the Mendelian concordance rate among trios and eliminates tens of thousands of spurious SNVs per sample, including a reduction of false positives in 269 challenging, medically relevant genes by up to a factor of 12. These corrections are in large part due to improvements to 70 protein-coding genes in >9 Mbp of inaccurate sequence caused by falsely collapsed or duplicated regions in GRCh38. Using the T2T-CHM13 genome also yields a more comprehensive view of SVs genome-wide, with a greatly improved balance of insertions and deletions. Finally, by providing numerous resources for T2T-CHM13 (including 1KGP genotypes, accessibility masks, and prominent annotation databases), our work will facilitate the transition to T2T-CHM13 from the current reference genome. CONCLUSION The vast improvements in variant discovery across samples of diverse ancestries position T2T-CHM13 to succeed as the next prevailing reference for human genetics. T2T-CHM13 thus offers a model for the construction and study of high-quality reference genomes from globally diverse individuals, such as is now being pursued through collaboration with the Human Pangenome Reference Consortium. As a foundation, our work underscores the benefits of an accurate and complete reference genome for revealing diversity across human populations. Genomic features and resources available for T2T-CHM13. Comparisons to GRCh38 reveal broad improvements in SNVs, indels, and SVs discovered across diverse human populations by means of short-read (1KGP) and long-read sequencing (LRS). These improvements are due to resolution of complex genomic loci (nonsyntenic and previously unresolved), duplication errors, and discordant haplotypes, including those in medically relevant genes.more » « less
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INTRODUCTION To faithfully distribute genetic material to daughter cells during cell division, spindle fibers must couple to DNA by means of a structure called the kinetochore, which assembles at each chromosome’s centromere. Human centromeres are located within large arrays of tandemly repeated DNA sequences known as alpha satellite (αSat), which often span millions of base pairs on each chromosome. Arrays of αSat are frequently surrounded by other types of tandem satellite repeats, which have poorly understood functions, along with nonrepetitive sequences, including transcribed genes. Previous genome sequencing efforts have been unable to generate complete assemblies of satellite-rich regions because of their scale and repetitive nature, limiting the ability to study their organization, variation, and function. RATIONALE Pericentromeric and centromeric (peri/centromeric) satellite DNA sequences have remained almost entirely missing from the assembled human reference genome for the past 20 years. Using a complete, telomere-to-telomere (T2T) assembly of a human genome, we developed and deployed tailored computational approaches to reveal the organization and evolutionary patterns of these satellite arrays at both large and small length scales. We also performed experiments to map precisely which αSat repeats interact with kinetochore proteins. Last, we compared peri/centromeric regions among multiple individuals to understand how these sequences vary across diverse genetic backgrounds. RESULTS Satellite repeats constitute 6.2% of the T2T-CHM13 genome assembly, with αSat representing the single largest component (2.8% of the genome). By studying the sequence relationships of αSat repeats in detail across each centromere, we found genome-wide evidence that human centromeres evolve through “layered expansions.” Specifically, distinct repetitive variants arise within each centromeric region and expand through mechanisms that resemble successive tandem duplications, whereas older flanking sequences shrink and diverge over time. We also revealed that the most recently expanded repeats within each αSat array are more likely to interact with the inner kinetochore protein Centromere Protein A (CENP-A), which coincides with regions of reduced CpG methylation. This suggests a strong relationship between local satellite repeat expansion, kinetochore positioning, and DNA hypomethylation. Furthermore, we uncovered large and unexpected structural rearrangements that affect multiple satellite repeat types, including active centromeric αSat arrays. Last, by comparing sequence information from nearly 1600 individuals’ X chromosomes, we observed that individuals with recent African ancestry possess the greatest genetic diversity in the region surrounding the centromere, which sometimes contains a predominantly African αSat sequence variant. CONCLUSION The genetic and epigenetic properties of centromeres are closely interwoven through evolution. These findings raise important questions about the specific molecular mechanisms responsible for the relationship between inner kinetochore proteins, DNA hypomethylation, and layered αSat expansions. Even more questions remain about the function and evolution of non-αSat repeats. To begin answering these questions, we have produced a comprehensive encyclopedia of peri/centromeric sequences in a human genome, and we demonstrated how these regions can be studied with modern genomic tools. Our work also illuminates the rich genetic variation hidden within these formerly missing regions of the genome, which may contribute to health and disease. This unexplored variation underlines the need for more T2T human genome assemblies from genetically diverse individuals. Gapless assemblies illuminate centromere evolution. ( Top ) The organization of peri/centromeric satellite repeats. ( Bottom left ) A schematic portraying (i) evidence for centromere evolution through layered expansions and (ii) the localization of inner-kinetochore proteins in the youngest, most recently expanded repeats, which coincide with a region of DNA hypomethylation. ( Bottom right ) An illustration of the global distribution of chrX centromere haplotypes, showing increased diversity in populations with recent African ancestry.more » « less
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null (Ed.)Alignments of multiple genomes are a cornerstone of comparative genomics, but generating these alignments remains technically challenging and often impractical. We developed the msa_pipeline workflow (https://bitbucket.org/bucklerlab/msa_pipeline) based on the LAST aligner to allow practical and sensitive multiple alignment of diverged plant genomes with minimal user inputs. Our workflow only requires a set of genomes in FASTA format as input. The workflow outputs multiple alignments in MAF format, and includes utilities to help calculate genome-wide conservation scores. As high repeat content and genomic divergence are substantial challenges in plant genome alignment, we also explored the impact of different masking approaches and alignment parameters using genome assemblies of 33 grass species. Compared to conventional masking with RepeatMasker, a k-mer masking approach increased the alignment rate of CDS and non-coding functional regions by 25% and 14% respectively. We further found that default alignment parameters generally perform well, but parameter tuning can increase the alignment rate for non-coding functional regions by over 52% compared to default LAST settings. Finally, by increasing alignment sensitivity from the default baseline, parameter tuning can increase the number of non-coding sites that can be scored for conservation by over 76%.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1186/s13059-022-02735-6
