Biology subjects

Miga, K. H.

Publications and source records attributed to Miga, K. H..

34 records · Page 2Linked to original sources

bioRxiv2021

From telomere to telomere: the transcriptional and epigenetic state of human repeat elements

Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.

genomics↗

bioRxiv2021

DiMeLo-seq: a long-read, single-molecule method for mappingprotein-DNA interactions genome-wide

Molecular studies of genome regulation often rely on the ability to map where specific proteins interact with genomic DNA. Existing techniques for mapping protein-DNA interactions genome-wide rely on DNA amplification methods followed by sequencing with short reads, which dissociates joint binding information at neighboring sites, removes endogenous DNA methylation information, and precludes the ability to reliably map interactions in repetitive regions of the genome. To address these limitations, we created a new protein-DNA mapping method, called Directed Methylation with Long-read sequencing (DiMeLo-seq), which methylates DNA near each target proteins DNA binding site in situ, then leverages the ability to distinguish methylated and unmethylated bases on long, native DNA molecules using long-read, single-molecule sequencing technologies. We demonstrate the optimization and utility of this method by mapping the interaction sites of a variety of different proteins and histone modifications across the human genome, achieving a single-molecule binding site resolution of less than 200 bp. Furthermore, we mapped the positions of the centromeric histone H3 variant CENP-A in repetitive regions that are unmappable with short reads, while simultaneously analyzing endogenous CpG methylation and joint binding events on single molecules. DiMeLo-seq is a versatile method that can provide multimodal and truly genome-wide information for investigating protein-DNA interactions.

genomics↗

bioRxiv2021

Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies

Advances in long-read sequencing technologies and genome assembly methods have enabled the recent completion of the first Telomere-to-Telomere (T2T) human genome assembly, which resolves complex segmental duplications and large tandem repeats, including centromeric satellite arrays in a complete hydatidiform mole (CHM13). Though derived from highly accurate sequencing, evaluation revealed that the initial T2T draft assembly had evidence of small errors and structural misassemblies. To correct these errors, we designed a novel repeat-aware polishing strategy that made accurate assembly corrections in large repeats without overcorrection, ultimately fixing 51% of the existing errors and improving the assembly QV to 73.9. By comparing our results to standard automated polishing tools, we outline common polishing errors and offer practical suggestions for genome projects with limited resources. We also show how sequencing biases in both PacBio HiFi and Oxford Nanopore Technologies reads cause signature assembly errors that can be corrected with a diverse panel of sequencing technologies

genomics↗

bioRxiv2021

Towards a Comprehensive Variation Benchmark for Challenging Medically-Relevant Autosomal Genes

The repetitive nature and complexity of multiple medically important genes make them intractable to accurate analysis, despite the maturity of short-read sequencing, resulting in a gap in clinical applications of genome sequencing. The Genome in a Bottle Consortium has provided benchmark variant sets, but these excluded some medically relevant genes due to their repetitiveness or polymorphic complexity. In this study, we characterize 273 of these 395 challenging autosomal genes that have multiple implications for medical sequencing. This extended, curated benchmark reports over 17,000 SNVs, 3,600 INDELs, and 200 SVs each for GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically important genes including CBS, CRYAA, and KCNE1. Our proposed solution improves variant recall in these genes from 8% to 100%. This benchmark will significantly improve the comprehensive characterization of these medically relevant genes and guide new method development.

genomics↗

bioRxiv2021

Epigenetic Patterns in a Complete Human Genome

The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, longrange epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.

genomics↗

bioRxiv2021

Segmental duplications and their variation in a complete human genome

Despite their importance in disease and evolution, highly identical segmental duplications (SDs) have been among the last regions of the human reference genome (GRCh38) to be finished. Based on a complete telomere-to-telomere human genome (T2T-CHM13), we present the first comprehensive view of human SD organization. SDs account for nearly one-third of the additional sequence increasing the genome-wide estimate from 5.4% to 7.0% (218 Mbp). An analysis of 266 human genomes shows that 91% of the new T2T-CHM13 SD sequence (68.3 Mbp) better represents human copy number. We find that SDs show increased single-nucleotide variation diversity when compared to unique regions; we characterize methylation signatures that correlate with duplicate gene transcription and predict 182 novel protein-coding gene candidates. We find that 63% (35.11/55.7 Mbp) of acrocentric chromosomes consist of SDs distinct from rDNA and satellite sequences. Acrocentric SDs are 1.75-fold longer (p=0.00034) than other SDs, are frequently shared with autosomal pericentromeric regions, and are heteromorphic among human chromosomes. Comparing long-read assemblies from other human (n=12) and nonhuman primate (n=5) genomes, we use the T2T-CHM13 genome to systematically reconstruct the evolution and structural haplotype diversity of biomedically relevant (LPA, SMN) and duplicated genes (TBC1D3, SRGAP2C, ARHGAP11B) important in the expansion of the human frontal cortex. The analysis reveals unprecedented patterns of structural heterozygosity and massive evolutionary differences in SD organization between humans and their closest living relatives.

genomics↗

bioRxiv2021

Haplotype-aware variant calling enables high accuracy in nanopore long-reads using deep neural networks

Long-read sequencing has the potential to transform variant detection by reaching currently difficult-to-map regions and routinely linking together adjacent variations to enable read based phasing. Third-generation nanopore sequence data has demonstrated a long read length, but current interpretation methods for its novel pore-based signal have unique error profiles, making accurate analysis challenging. Here, we introduce a haplotype-aware variant calling pipeline PEPPER-Margin-DeepVariant that produces state-of-the-art variant calling results with nanopore data. We show that our nanopore-based method outperforms the short-read-based single nucleotide variant identification method at the whole genome-scale and produces high-quality single nucleotide variants in segmental duplications and low-mappability regions where short-read based genotyping fails. We show that our pipeline can provide highly-contiguous phase blocks across the genome with nanopore reads, contiguously spanning between 85% to 92% of annotated genes across six samples. We also extend PEPPER-Margin-DeepVariant to PacBio HiFi data, providing an efficient solution with superior performance than the current WhatsHap-DeepVariant standard. Finally, we demonstrate de novo assembly polishing methods that use nanopore and PacBio HiFi reads to produce diploid assemblies with high accuracy (Q35+ nanopore-polished and Q40+ PacBio-HiFi-polished).

bioinformatics↗

bioRxiv2020

The structure, function, and evolution of a complete human chromosome 8

The complete assembly of each human chromosome is essential for understanding human biology and evolution. Using complementary long-read sequencing technologies, we complete the first linear assembly of a human autosome, chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08 Mbp centromeric -satellite array, a 644 kbp defensin copy number polymorphism important for disease risk, and an 863 kbp variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric -satellite array is generally methylated except for a 73 kbp hypomethylated region of diverse higher-order -satellite enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. Using a dual long-read sequencing approach, we complete the assembly of the orthologous chromosome 8 centromeric regions in chimpanzee, orangutan, and macaque for the first time to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order -satellite structure evolved specifically in the great ape ancestor, and the centromeric region evolved with a layered symmetry, with more ancient higher-order repeats located at the periphery adjacent to monomeric -satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated at least 2.2-fold, and this acceleration extends beyond the higher-order -satellite into the flanking sequence.

genomics↗

bioRxiv2020

Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations

Centromeres play a fundamental role in chromosome segregation. Although originally thought to be silent chromosomal regions, centromeres are actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci fluctuate in their levels over the cell cycle and do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Our results demonstrate that alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require centromere proteins and other cell division components. Instead, our work implicates centromere-nucleolar associations as the major factor regulating alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels, explaining variations in alpha-satellite RNA between cell lines. In addition, alpha-satellite transcript levels increase substantially when the nucleolus is disrupted. Together, our results are inconsistent with a direct, physical role for alpha-satellite transcripts in cell division processes, and instead support a role for ongoing transcription in promoting centromere chromatin dynamics. The control of alpha-satellite transcription by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

cell biology↗

bioRxiv2020

HiCanu: accurate assembly of segmental duplications, satellites, and allelic variants from high-fidelity long reads

Complete and accurate genome assemblies form the basis of most downstream genomic analyses and are of critical importance. Recent genome assembly projects have relied on a combination of noisy long-read sequencing and accurate short-read sequencing, with the former offering greater assembly continuity and the latter providing higher consensus accuracy. The recently introduced PacBio HiFi sequencing technology bridges this divide by delivering long reads (>10 kbp) with high per-base accuracy (>99.9%). Here we present HiCanu, a significant modification of the Canu assembler designed to leverage the full potential of HiFi reads via homopolymer compression, overlap-based error correction, and aggressive false overlap filtering. We benchmark HiCanu with a focus on the recovery of haplotype diversity, major histocompatibility complex (MHC) variants, satellite DNAs, and segmental duplications. For diploid human genomes sequenced to 30x HiFi coverage, HiCanu achieved superior accuracy and allele recovery compared to the current state of the art. On the effectively haploid CHM13 human cell line, HiCanu achieved an NG50 contig size of 77 Mbp with a per-base consensus accuracy of 99.999% (QV50), surpassing recent assemblies of high-coverage, ultra-long Oxford Nanopore reads in terms of both accuracy and continuity. This HiCanu assembly correctly resolves 337 out of 341 validation BACs sampled from known segmental duplications and provides the first preliminary assemblies of 9 complete human centromeric regions. Although gaps and errors still remain within the most challenging regions of the genome, these results represent a significant advance towards the complete assembly of human genomes. AvailabilityHiCanu is implemented within the Canu assembly framework and is available from https://github.com/marbl/canu.

bioinformatics↗

bioRxiv2019

TandemMapper and TandemQUAST: mapping long reads and assessing/improving assembly quality in extra-long tandem repeats

Extra-long tandem repeats (ETRs) are widespread in eukaryotic genomes and play an important role in fundamental cellular processes, such as chromosome segregation. Although emerging long-read technologies have enabled ETR assemblies, the accuracy of such assemblies is difficult to evaluate since there is no standard tool for their quality assessment. Moreover, since the mapping of long error-prone reads to ETR remains an open problem, it is not clear how to polish draft ETR assemblies. To address these problems, we developed the tandemMapper tool for mapping reads to ETRs and the tandemQUAST tool for polishing ETR assemblies and their quality assessment. We demonstrate that tandemQUAST not only reveals errors in and evaluates ETR assemblies, but also improves them. To illustrate how tandemMapper and tandemQUAST work, we apply them to recently generated assemblies of human centromeres.

bioinformatics↗

bioRxiv2019

Telomere-to-telomere assembly of a complete human X chromosome

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the [~]2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

bioinformatics↗

bioRxiv2019

Efficient de novo assembly of eleven human genomes using PromethION sequencing and a novel nanopore toolkit

Present workflows for producing human genome assemblies from long-read technologies have cost and production time bottlenecks that prohibit efficient scaling to large cohorts. We demonstrate an optimized PromethION nanopore sequencing method for eleven human genomes. The sequencing, performed on one machine in nine days, achieved an average 63x coverage, 42 Kb read N50, 90% median read identity and 6.5x coverage in 100 Kb+ reads using just three flow cells per sample. To assemble these data we introduce new computational tools: Shasta - a de novo long read assembler, and MarginPolish & HELEN - a suite of nanopore assembly polishing algorithms. On a single commercial compute node Shasta can produce a complete human genome assembly in under six hours, and MarginPolish & HELEN can polish the result in just over a day, achieving 99.9% identity (QV30) for haploid samples from nanopore reads alone. We evaluate assembly performance for diploid, haploid and trio-binned human samples in terms of accuracy, cost, and time and demonstrate improvements relative to current state-of-the-art methods in all areas. We further show that addition of proximity ligation (Hi-C) sequencing yields near chromosome-level scaffolds for all eleven genomes.

bioinformatics↗

bioRxiv2019

Chromosomal rearrangements at hypomethylated Satellite 2 sequences are associated with impaired replication efficiency and increased fork stalling

Cancer cells, aging cells, and cells from patients with the developmental disorder Immunodeficiency, Centromeric instability, and Facial anomalies (ICF) syndrome frequently display a striking loss of DNA methylation (hypomethylation) that is accompanied by increased DNA damage and chromosomal rearrangements. Despite the robust link, the mechanism by which hypomethylation leads to genomic instability is poorly understood. We report that the human pericentromeric repeat sequence Satellite 2 (SAT2) poses challenges to the DNA replication machinery when hypomethylated. Loss of methylation at SAT2 is associated with increased frequencies of chromosomal abnormalities and DNA damage. Hypomethylation of SAT2 is associated with elevated levels of replication stress signaling, and chromosomal abnormalities involving SAT2 are enhanced by low levels of aphidicolin-induced replication stress. To investigate the basis for these chromosomal abnormalities, we developed a single-molecule approach employing DNA combing to examine the progress of replication forks through SAT2 at the resolution of a single DNA molecule. Our analysis of replicating SAT2 molecules provides in vivo evidence that hypomethylation of SAT2 strongly decreases the efficiency of replicating these sequences suggesting that hypomethylation results in the formation of barriers to the replication machinery. Consistent with increased frequency of fork stalling at these sequences, we find increased levels of single-stranded DNA (ssDNA) binding protein RPA2 as well as asymmetric progression of sister replication forks within hypomethylated SAT2 sequences. Together these findings indicate that impaired replication triggers the formation of chromosomal aberrations observed at hypomethylated SAT2 sequences and also suggests a mechanistic basis for how the loss of DNA methylation may contribute to genomic instability in diverse pathological conditions.

molecular biology↗

bioRxiv2017

Linear Assembly of a Human Y Centromere using Nanopore Long Reads

The human genome reference sequence remains incomplete due to the challenge of assembling long tracts of near-identical tandem repeats-in centromeric regions. To address this, we have implemented a nanopore sequencing strategy to generate high quality reads that span hundreds of kilobases of highly repetitive DNAs. Here, we use this advance to produce a sequence assembly and characterization of the centromeric region of a human Y chromosome.

genomics↗

bioRxiv2017

Nanopore sequencing and assembly of a human genome with ultra-long reads

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data ([~]30x theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 [~]3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5x-coverage of ultra-long reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.

genomics↗