Biology subjects

Grigorev, K.

Publications and source records attributed to Grigorev, K..

5 recordsLinked to original sources

bioRxiv2024

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map ([~]3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

genomics↗

bioRxiv2020

Engineering Radioprotective Human Cells Using the Tardigrade Damage Suppressor Protein, DSUP

Spaceflight has been documented to produce detrimental effects to physiology and genomic stability, partly a result of Galactic Cosmic Radiation (GCR). In recent years, extensive research into extremotolerant organisms has begun to reveal how they survive harsh conditions, such as ionizing radiation. One such organism is the tardigrade (Ramazzottius varieornatus) which can survive up to 5kGy of ionizing radiation and the vacuum of space. In addition to their extensive network of DNA damage response mechanisms, the tardigrade also possesses a unique damage suppressor protein (Dsup) that co-localizes with chromatin in both tardigrade and transduced human cells to protect against DNA damage from reactive oxygen species induced by ionizing radiation. While Dsup has been shown to confer human cells with increased radiotolerance; much of the mechanism of how it does this in the context of human cells remains unknown. Until now there is no knowledge yet of how introduction of Dsup into human cells can perturb molecular networks and if there are any systemic risks associated with foreign gene introduction. Here, we created a stable HEK293 cell line expressing Dsup, validated its radioprotective phenotype, and performed multi-omic analyses across different time points and doses of radiation to delineate molecular mechanism of the radioprotection and assess molecular network pertubations. Dsup expressing human cells showed an enrichment for pathways seen in cells overexpressing HMGN1, a chromosomal architectural protein that has a highly similar nucleosome binding motif. As HMGN1 binding to nucleosomes promotes a less transcriptionally repressed chromatin state, we further explored the hypothesis that Dsup could behave similarly via ATAC-seq analysis and discovered overall selective differential opening and closing of the chromatin landscape. Cut&Run analysis further revealed global increases in histone post translational modifications indicative of open chromatin and global decreases in repressive marks, with Dsup binding preferentially towards promoter regions marked by H3K27ac and H3K4me3. We further validated some of the enriched pathways via in-vitro assays and revealed novel phenotypes that Dsup confers to human cells such as reduction in apoptosis, increased cell proliferation, and increased cell adhesion properties. Our analysis provides evidence that the Dsup protein in the context of HEK293 cells may behave as a chromatin architectural protein and that in addition to its nucleosome shielding effect, may confer radio-resistance via chromatin modulation. These results provide future insight into mitigating some of the major challenges involved with long term spaceflight as well as understanding some of the molecular architectural underpinnings that lead to radioresistant cancer phenotypes back home.

synthetic biology↗

bioRxiv2020

Cell-free DNA (cfDNA) and exosome profiling from a year-long humanspaceflight reveals circulating biomarkers

The health impact of prolonged space flight on the human body is not well understood. Liquid biopsies based on cell-free DNA (cfDNA) or exosome analysis provide a noninvasive approach to monitor the dynamics of genomic, epigenomic and proteomic biomarkers, and the occurrence of DNA damage, physiological stress, and immune responses. To study the molecular consequences of spaceflight we profiled cfDNA isolated from plasma of an astronaut (TW) during a year-long mission on the International Space Station (ISS), sampling before, during, and after spaceflight, and compared the results to cfDNA profiling of the subjects identical twin (HR) who remained on Earth, as well as healthy donors. We characterized cfDNA concentration and fragment size, and the positioning of nucleosomes on cfDNA, observing a significant increase in the proportion of cell-free mitochondrial DNA inflight, suggesting that cf-mtDNA is a potential biomarker for space flight-associated stress, and that this result was robust to ambient transit from the International Space Station (ISS). Analysis of exosomes isolated from post-flight plasma revealed a 30-fold increase in circulating exosomes and distinct exosomal protein cargo, including brain-derived peptides, in TW compared to HR and all known controls. This study provides the first longitudinal analysis of astronaut cfDNA during spaceflight, as well as the first exosome profiles, and highlights cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and other unique environmental conditions on the ISS.

cell biology↗

bioRxiv2020

Haplotype Diversity and Sequence Heterogeneity of Human Telomeres

Telomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, whose lengths can be correlated with age and disease risk factors. Given their length and repetitive nature, telomeric regions are not easily reconstructed from short read sequencing, making telomere sequence resolution a very costly and generally intractable problem. Recently, long-read sequencing, with read lengths measuring in hundreds of Kbp, has made it possible to routinely read into telomeric regions and inspect their structure. Here, we describe a framework for extracting telomeric reads from single-molecule sequencing experiments, describing their sequence variation and motifs, and for haplotype inference. We find that long telomeric stretches can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize non-canonical motifs (both previously reported as well as novel), and report the first motif composition maps of human telomeric diplotypes on a multi-Kbp scale.

genomics↗

bioRxiv2017

Innovative assembly strategy contributes to the understanding of evolution and conservation genetics of the critically endangered Solenodon paradoxus from the island of Hispaniola

Solenodons are insectivores living on the Caribbean islands, with few surviving related taxa. The genus occupies one of the most ancient branches among the placental mammals. The history, unique biology and adaptations of these enigmatic venomous species, can be greatly advanced given the availability of genome data, but the whole genome assembly for solenodons has never been previously performed, partially due to the difficulty in obtaining samples from the field. Island isolation has likely resulted in extreme homozygosity within the Hispaniolan solenodon (Solenodon paradoxus), thus we tested the performance of several assembly strategies for performance with genetically impoverished species genomes. The string-graph based assembly strategy seems a better choice compared to the conventional de Brujn graph approach, due to the high levels of homozygosity, which is often a hallmark of endemic or endangered species. A consensus reference genome was assembled from sequences of five individuals from the southern subspecies (S. p. woodi). In addition, we obtained one additional sequence of the northern subspecies (S. p. paradoxus). The resulting genome assemblies were compared to each other, and annotated for genes, with a specific emphasis on the venomous genes, repeats, variable microsatellite loci and other genomic variants. Phylogenetic positioning and selection signatures were inferred based on 4,416 single copy orthologs from 10 other mammals. Patterns of SNP variation allowed us to infer population demography, which indicated a subspecies split within the Hispaniolan solenodon at least 300 Kya.

evolutionary biology↗