Biology subjects

Gershman, A.

Publications and source records attributed to Gershman, A..

10 recordsLinked to original sources

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

Genomic insights into metabolic flux in ruby-throated hummingbirds

Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome level de novo genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long and short read sequencing and scaffolding using other existing assemblies. We then used hybrid long and short-read RNA-sequencing for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and a deletion of critical genes (GLUT4, GCK) involved in glucostasis in other vertebrates. We found expression of fructose-specific GLUT5 putatively in place of insulin-sensitive GLUT4, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds suggesting key pathways for the rapid metabolic switch hummingbirds undergo.

genomics↗

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

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

Chromosome specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing

We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome specific telomere lengths may be explained by stochastic clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosomes end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y sequences, tel1{Delta} rif1{Delta} double mutants had a very small, but significant, increase in telomere length compared to the tel1{Delta} single mutant, suggesting an influence of Y elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2{Delta}) and found the minimal telomere length to be around 75bp. In these est2{Delta} mutants there were many apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2{Delta} rad52{Delta} double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, suggests possible new biological mechanisms regulating telomere length, and provides capability to test new hypotheses.

molecular biology↗

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

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↗

bioRxiv2020

De novo genome assembly of the Tobacco Hornworm moth (Manduca sexta)

The Tobacco hornworm, Manduca sexta, is a lepidopteran insect that is used extensively as a model system for studying insect biology, development, neuroscience and immunity. However, current studies rely on the highly fragmented reference genome Msex_1.0, which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. We present the new reference genome for M. sexta, JHU_Msex_v1.0, applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly is 470 Mb and is ~20x more continuous than the original assembly, with scaffold N50 >14 Mb. We annotated the assembly by lifting over existing annotations and supplementing with additional supporting RNA-based data for a total of 25,256 genes. The new reference assembly is accessible in annotated form for public use. We demonstrate that improved continuity of the M. sexta genome improves resequencing studies and benefits future research on M. sexta as a model organism.

genomics↗

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↗