Biology subjects

Rhie, A.

Publications and source records attributed to Rhie, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation

Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the NIH Center for Alzheimers and Related Dementias (CARD). Using a single PromethION flow cell, we can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains difficult within homopolymers and tandem repeats, but is comparable to Illumina calls elsewhere. Further, we can discover structural variants with F1-score comparable to state-of-the-art methods involving Pacific Biosciences HiFi sequencing and trio information (but at a lower cost and greater throughput). Using ONT-based phasing, we can then combine and phase small and structural variants at megabase scales. Our protocol also produces highly accurate, haplotype-specific methylation calls. Overall, this makes large-scale long-read sequencing projects feasible; the protocol is currently being used to sequence thousands of brain-based genomes as a part of the NIH CARD initiative. We provide the protocol and software as open-source integrated pipelines for generating phased variant calls and assemblies.

bioinformatics↗

bioRxiv2022

The complete sequence of a human Y chromosome

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure including long palindromes, tandem repeats, and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4, 5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029 base pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, revealing the complete ampliconic structures of TSPY, DAZ, and RBMY gene families; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a prior assembly of the CHM13 genome4 and mapped available population variation, clinical variants, and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

genomics↗

bioRxiv2022

Recombination between heterologous human acrocentric chromosomes

The short arms of the human acrocentric chromosomes 13, 14, 15, 21, and 22 share large homologous regions, including the ribosomal DNA repeats and extended segmental duplications (Floutsakou et al. 2013; van Sluis et al. 2019). While the complete assembly of these regions in the Telomere-to-Telomere consortiums CHM13 provided a model of their homology (Nurk et al. 2022), it remained unclear if these patterns were ancestral or maintained by ongoing recombination exchange. Here, we show that acrocentric chromosomes contain pseudo-homologous regions (PHRs) indicative of recombination between non-homologs. Considering an all-to-all comparison of the high-quality human pangenome from the Human Pangenome Reference Consortium (HPRC) (Liao et al. 2022), we find that contigs from all of the acrocentric short arms form a community similar to those formed by single chromosomes or the sex chromosome pair. A variation graph (Garrison et al. 2018) constructed from centromere-spanning acrocentric contigs indicates the presence of regions where most contigs appear nearly identical between heterologous CHM13 acrocentrics. Except on chromosome 15, we observe faster decay of linkage disequilibrium in the PHRs than in the corresponding short and long arms, indicating higher rates of recombination (N. Li and Stephens 2003; Huttley et al. 1999). The PHRs include sequences previously shown to lie at the breakpoint of Robertsonian translocations (Jarmuz-Szymczak et al. 2014), and we show that their arrangement is compatible with crossover in inverted duplications on chromosomes 13, 14, and 21. The ubiquity of signals of recombination between heterologous chromosomes seen in the HPRC draft pangenomes acrocentric assemblies suggests that these shared sequences form the basis for recurrent Robertsonian translocations, providing sequence and population-based confirmation of hypotheses first developed cytogenetically fifty years ago (Hamerton et al. 1975).

genomics↗

bioRxiv2022

Verkko: telomere-to-telomere assembly of diploid chromosomes

The Telomere-to-Telomere consortium recently assembled the first truly complete sequence of a human genome. To resolve the most complex repeats, this project relied on manual integration of ultra-long Oxford Nanopore sequencing reads with a high-resolution assembly graph built from long, accurate PacBio HiFi reads. We have improved and automated this strategy in Verkko, an iterative, graph-based pipeline for assembling complete, diploid genomes. Verkko begins with a multiplex de Bruijn graph built from long, accurate reads and progressively simplifies this graph via the integration of ultra-long reads and haplotype-specific markers. The result is a phased, diploid assembly of both haplotypes, with many chromosomes automatically assembled from telomere to telomere. Running Verkko on the HG002 human genome resulted in 20 of 46 diploid chromosomes assembled without gaps at 99.9997% accuracy. The complete assembly of diploid genomes is a critical step towards the construction of comprehensive pangenome databases and chromosome-scale comparative genomics.

bioinformatics↗

bioRxiv2022

Pangenomics provides insights into the role of synanthropy in barn swallow evolution

Insights into the evolution of non-model organisms are often limited by the lack of reference genomes. As part of the Vertebrate Genomes Project, we present a new reference genome and a pangenome produced with High-Fidelity long reads for the barn swallow Hirundo rustica. We then generated a reference-free multialignment with other bird genomes to identify genes under selection. Conservation analyses pointed at genes enriched for transcriptional regulation and neurodevelopment. The most conserved gene is CAMK2N2, with a potential role in fear memory formation. In addition, using all publicly available data, we generated a comprehensive catalogue of genetic markers. Genome-wide linkage disequilibrium scans identified potential selection signatures at multiple loci. The top candidate region comprises several genes and includes BDNF, a gene involved in stress response, fear memory formation, and tameness. We propose that the strict association with humans in this species is linked with the evolution of pathways typically under selection in domesticated taxa.

genomics↗

bioRxiv2022

Automated assembly of high-quality diploid human reference genomes

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has greatly benefited society1, 2. However, it still has many gaps and errors, and does not represent a biological human genome since it is a blend of multiple individuals3, 4. Recently, a high-quality telomere-to-telomere reference genome, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a duplicate genome, and is thus nearly homozygous5. To address these limitations, the Human Pangenome Reference Consortium (HPRC) recently formed with the goal of creating a collection of high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and automated assembly approaches yields the most complete, accurate, and cost-effective diploid genome assemblies with minimal manual curation. Approaches that used highly accurate long reads and parent-child data to sort haplotypes during assembly outperformed those that did not. Developing a combination of all the top performing methods, we generated our first high- quality diploid reference assembly, containing only [~]4 gaps (range 0-12) per chromosome, most within + 1% of CHM13s length. Nearly 1/4th of protein coding genes have synonymous amino acid changes between haplotypes, and centromeric regions showed the highest density of variation. Our findings serve as a foundation for assembling near-complete diploid human genomes at the scale required for constructing a human pangenome reference that captures all genetic variation from single nucleotides to large structural rearrangements.

genomics↗

bioRxiv2021

Recombination suppression and selection affect local ancestries in genomes of a migratory songbird

Genetic variation of the entire genome represents population structure, yet individual loci can show distinct patterns. Such deviations identified through genome scans have often been attributed to effects of selection instead of randomness. This interpretation assumes that long enough genomic intervals average out randomness in underlying genealogies, which represent local genetic ancestries. However, an alternative explanation to distinct patterns has not been fully addressed: too few genealogies to average out the effect of randomness. Specifically, distinct patterns of genetic variation may be due to reduced local recombination rate, which reduces the number of genealogies in a genomic window. Here, we associate distinct patterns of local genetic variation with reduced recombination rates in a songbird, the Eurasian blackcap (Sylvia atricapilla), using genome sequences and recombination maps. We find that distinct patterns of local genetic variation reflect haplotype structure at low-recombining regions either shared in most populations or found only in a few populations. At the former species-wide low-recombining regions, genetic variation depicts conspicuous haplotypes segregating in multiple populations. At the latter population-specific low-recombining regions, genetic variation represents variance among cryptic haplotypes within the low-recombining populations. With simulations, we confirm that these distinct patterns of haplotype structure evolve due to reduced recombination rate, on which the effects of selection can be overlaid. Our results highlight that distinct patterns of genetic variation can emerge through evolution of reduced local recombination rate. Recombination landscape as an evolvable trait therefore plays an important role determining the heterogeneous distribution of genetic variation along the genome.

evolutionary biology↗

bioRxiv2021

A haplotype-resolved genome assembly of the Nile rat facilitates exploration of the genetic basis of diabetes

The Nile rat (Avicanthis niloticus) is an important animal model for biomedical research, including the study of diurnal rhythms and type 2 diabetes. Here, we report a 2.5 Gb, chromosome-level reference genome assembly with fully resolved parental haplotypes, generated with the Vertebrate Genomes Project (VGP). The assembly is highly contiguous, with contig N50 of 11.1 Mb, scaffold N50 of 83 Mb, and 95.2% of the sequence assigned to chromosomes. We used a novel workflow to identify 3,613 segmental duplications and quantify duplicated genes. Comparative analyses revealed unique genomic features of the Nile rat, including those that affect genes associated with type 2 diabetes and metabolic dysfunctions. These include 14 genes that are heterozygous in the Nile rat or highly diverged from the house mouse. Our findings reflect the exceptional level of genomic detail present in this assembly, which will greatly expand the potential of the Nile rat as a model organism for genetic studies.

genomics↗

bioRxiv2021

Merfin: improved variant filtering and polishing via k-mer validation

Read mapping and variant calling approaches have been widely used for accurate genotyping and improving consensus quality assembled from noisy long reads. Variant calling accuracy relies heavily on the read quality, the precision of the read mapping algorithm and variant caller, and the criteria adopted to filter the calls. However, it is impossible to define a single set of optimal parameters, as they vary depending on the quality of the read set, the variant caller of choice, and the quality of the unpolished assembly. To overcome this issue, we have devised a new tool called Merfin (k-mer based finishing tool), a k-mer based variant filtering algorithm for improved genotyping and polishing. Merfin evaluates the accuracy of a call based on expected k-mer multiplicity in the reads, independently of the quality of the read alignment and variant callers internal score. Moreover, we introduce novel assembly quality and completeness metrics that account for the expected genomic copy numbers. Merfin significantly increased the precision of a variant call and reduced frameshift errors when applied to PacBio HiFi, PacBio CLR, or Nanopore long read based assemblies. We demonstrate the utility while polishing the first complete human genome, a fully phased human genome, and non-human high-quality genomes.

bioinformatics↗

bioRxiv2021

Complete genomic and epigenetic maps of human centromeres

Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics. One-sentence summaryDeep characterization of fully assembled human centromeres reveals their architecture and fine-scale organization, variation, and evolution.

genomics↗

bioRxiv2021

A complete reference genome improves analysis of human genetic variation

Compared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 Mbp of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome to clinical and functional study. Here we demonstrate how the new reference universally improves read mapping and variant calling for 3,202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of novel variants per sample--a new frontier for evolutionary and biomedical discovery. Simultaneously, the new reference eliminates tens of thousands of spurious variants per sample, including up to 12-fold reduction of false positives in 269 medically relevant genes. The vast improvement in variant discovery coupled with population and functional genomic resources position T2T-CHM13 to replace GRCh38 as the prevailing reference for human genetics. One Sentence SummaryThe T2T-CHM13 reference genome universally improves the analysis of human genetic variation.

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

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

The complete sequence of a human genome

In 2001, Celera Genomics and the International Human Genome Sequencing Consortium published their initial drafts of the human genome, which revolutionized the field of genomics. While these drafts and the updates that followed effectively covered the euchromatic fraction of the genome, the heterochromatin and many other complex regions were left unfinished or erroneous. Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release. The new T2T-CHM13 reference includes gapless assemblies for all 22 autosomes plus Chromosome X, corrects numerous errors, and introduces nearly 200 million bp of novel sequence containing 2,226 paralogous gene copies, 115 of which are predicted to be protein coding. The newly completed regions include all centromeric satellite arrays and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies for the first time.

genomics↗

bioRxiv2021

False gene and chromosome losses affected by assembly and sequence errors

Many genome assemblies have been found to be incomplete and contain misassemblies. The Vertebrate Genomes Project (VGP) has been producing assemblies with an emphasis on being as complete and error-free as possible, utilizing long reads, long-range scaffolding data, new assembly algorithms, and manual curation. Here we evaluate these new vertebrate genome assemblies relative to the previous references for the same species, including a mammal (platypus), two birds (zebra finch, Annas hummingbird), and a fish (climbing perch). We found that 3 to 11% of genomic sequence was entirely missing in the previous reference assemblies, which included nearly entire GC-rich and repeat-rich microchromosomes with high gene density. Genome-wide, between 25 to 60% of the genes were either completely or partially missing in the previous assemblies, and this was in part due to a bias in GC-rich 5-proximal promoters and 5 exon regions. Our findings reveal novel regulatory landscapes and protein coding sequences that have been greatly underestimated in previous assemblies and are now present in the VGP assemblies.

genomics↗

bioRxiv2021

Widespread false gene gains caused by duplication errors in genome assemblies

False duplications in genome assemblies lead to false biological conclusions. We quantified false duplications in previous genome assemblies and their new counterparts of the same species (platypus, zebra finch, Annas hummingbird) generated by the Vertebrate Genomes Project (VGP). Whole genome alignments revealed that 4 to 16% of the sequences were falsely duplicated in the previous assemblies, impacting hundreds to thousands of genes. These led to overestimated gene family expansions. The main source of the false duplications was heterotype duplications, where the haplotype sequences were more divergent than other parts of the genome leading the assembly algorithms to classify them as separate genes or genomic regions. A minor source was sequencing errors. Although present in a smaller proportion, we observed false duplications remaining in the VGP assemblies that can be identified and purged. This study highlights the need for more advanced assembly methods that better separates haplotypes and sequence errors, and the need for cautious analyses on gene gains.

genomics↗

bioRxiv2020

A long read mapping method for highly repetitive reference sequences

About 5-10% of the human genome remains inaccessible for functional analysis due to the presence of repetitive sequences such as segmental duplications and tandem repeat arrays. To enable high-quality resequencing of personal genomes, it is crucial to support end-to-end genome variant discovery using repeat-aware read mapping methods. In this study, we highlight the fact that existing long read mappers often yield incorrect alignments and variant calls within long, near-identical repeats, as they remain vulnerable to allelic bias. In the presence of a non-reference allele within a repeat, a read sampled from that region could be mapped to an incorrect repeat copy because the standard pairwise sequence alignment scoring system penalizes true variants. To address the above problem, we propose a novel, long read mapping method that addresses allelic bias by making use of minimal confidently alignable substrings (MCASs). MCASs are formulated as minimal length substrings of a read that have unique alignments to a reference locus with sufficient mapping confidence (i.e., a mapping quality score above a user-specified threshold). This approach treats each read mapping as a collection of confident sub-alignments, which is more tolerant of structural variation and more sensitive to paralog-specific variants (PSVs) within repeats. We mathematically define MCASs and discuss an exact algorithm as well as a practical heuristic to compute them. The proposed method, referred to as Winnowmap2, is evaluated using simulated as well as real long read benchmarks using the recently completed gapless assemblies of human chromosomes X and 8 as a reference. We show that Winnowmap2 successfully addresses the issue of allelic bias, enabling more accurate downstream variant calls in repetitive sequences. As an example, using simulated PacBio HiFi reads and structural variants in chromosome 8, Winnowmap2 alignments achieved the lowest false-negative and false-positive rates (1.89%, 1.89%) for calling structural variants within near-identical repeats compared to minimap2 (39.62%, 5.88%) and NGMLR (56.60%, 36.11%) respectively. Winnowmap2 code is accessible at https://github.com/marbl/Winnowmap

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↗