Specific Structural Variants Research Articles

Genome variation and LTR-RT analyses of an ancient peach landrace reveal mechanism of blood-flesh fruit color formation and fruit maturity date advancement.

Peach (Prunus persica) landrace has typical regional characteristics, strong environmental adaptability, and contains many valuable genes that provide the foundation for breeding excellent varieties. Therefore, it is necessary to assemble the genomes of specific landraces to facilitate the localization and utilization of these genes. Here, we de novo assembled a high-quality genome from an ancient blood-fleshed Chinese landrace Tianjin ShuiMi (TJSM) that originated from the China North Plain. The assembled genome size was 243.5Mb with a contig N50 of 23.7Mb and a scaffold N50 of 28.6Mb. Compared with the reported peach genomes, our assembled TJSM genome had the largest number of specific structural variants (SVs) and long terminal repeat-retrotransposons (LTR-RTs). Among the LTR-RTs with the potential to regulate their host genes, we identified a 6688bp LTR-RT (named it blood TE) in the promoter of NAC transcription factor-encoding PpBL, a gene regulating peach blood-flesh formation. The blood TE was not only co-separated with the blood-flesh phenotype but also associated with fruit maturity date advancement and different intensities of blood-flesh color formation. Our findings provide new insights into the mechanism underlying the development of the blood-flesh color and determination of fruit maturity date and highlight the potential of the TJSM genome to mine more variations related to agronomic traits in peach fruit.

Solving the unsolved genetic epilepsies - current and future perspectives.

Many patients suffering from epilepsy undergo exome or genome sequencing as part of a diagnostic work-up, however, many remain genetically unsolved. There are various factors that account for negative results in exome/genome sequencing for epilepsy patients; 1) the underlying cause is not genetic, 2) there is a complex polygenic explanation, 3) the illness is monogenic but the causative gene remains to be linked to a human disorder, 4) family segregation with reduced penetrance, 5) somatic mosaicism or the complexity of eg. a structural rearrangement, or 6) limited knowledge or diagnostic tools hinder the proper classification of a variant, resulting in its designation as a variant of unknown significance. The objective of this review is to outline some of the diagnostic options that lie beyond the exome/genome, and that might become clinically relevant within the foreseeable future. These options include: 1) re-analysis of older exome/genome data as knowledge increases or symptoms change, 2) looking for somatic mosaicism or long-read sequencing to detect low-complexity repeat variants or specific structural variants missed by traditional exome/genome sequencing, 3) exploration of the non-coding genome including disruption of topologically associated domains, long range non-coding RNA or other regulatory elements, and finally 4) transcriptomics, DNA methylation signatures and metabolomics as complementary diagnostic methods that may be used in the assessment of variants of unknown significance. Some of these tools are currently not integrated into standard diagnostic work-up. However, it is reasonable to expect that they will become increasingly available and improve current diagnostic capabilities, thereby enabling precision diagnosis in patients that are currently undiagnosed.

Abstract LB081: Concurrent characterization of somatic nucleotide mutations, 5mC, and 5hmC methylation in clinical research samples using a single nanopore sequencing assay

Abstract Genomic and epigenomic variants accumulate during cancer progression, resulting in a tumor consisting of heterogeneous collections of cells, or clones, with distinct profiles of genomic and/or epigenomic alterations. The ability to characterize a broad set of somatic (epi)genomic variations simultaneously, using a single sequencing assay, facilitates cancer research and can help inform treatment. Here we describe a comprehensive approach to analyze native tumor DNA with long nanopore reads. First, we demonstrate that shallow (<1x) whole genome nanopore sequencing provides sufficient information to observe large-scale copy number aberrations. This enables rapid and affordable screening of samples with an aim of selecting those that contain variation(s) of potential interest; such samples can then be sequenced to higher coverages for a higher resolution picture of specific structural variants. We further demonstrate an analysis workflow vimba for high coverage (>30x) nanopore sequencing of tumor/normal datasets that detects clone- and haplotype-resolved structural variations in patient-derived research samples. We provide an example of haplotype- and breakpoint-resolved compounded amplification and loss of heterozygosity (LOH) of a region containing the EGFR gene in an admixed kidney tumor sample. Finally, we describe the suitability of this assay to gather genome-wide (epi)genomic variations (e.g., SNPs, indels, SVs, microsatellite instability) across tumor samples of different cancer types. For the first time with native long nanopore reads we are able to capture 5mC and 5hmC methylation information and map it over some of the most challenging repetitive regions of the genome. This information can empower a better understanding of cancer-type-specific (epi)genomic alterations, which in turn can help map out the landscape for further research. Our approach demonstrates how nanopore sequencing can be used as a single comprehensive platform to better understand and resolve the complexity of (epi)genomic somatic alterations in tumor samples. Citation Format: Sergey Aganezov, Philipp Rescheneder, John Beaulaurier, Phillip James, Sissel Juul. Concurrent characterization of somatic nucleotide mutations, 5mC, and 5hmC methylation in clinical research samples using a single nanopore sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB081.

High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

Planigraphic (Spatial) Distribution of Mitochondrial DNA Variants at the Andronovo Time Cemetery Tartas-1: Preliminary Results

The most informative funerary site documenting the Andronovo (Fedorovo) migration to the West Siberian forest-steppe is Tartas-1, where more than 600 burials of that period, associated with Late Krotovo (Cherno- Ozerye) and Andronovo (Fedorovo) cultures, have been excavated to date. On the basis of skeletal remains from these burials, we have formed and successfully analyzed 256 samples of mitochondrial DNA, uniformly covering the entire area of the cemetery. This approach is a genetic counterpart of the continuous area method, which is used to excavate Tartas-1. The article opens a series of publications addressing the analysis and interpretation of this sample. We present the findings of the first stage in the analysis of spatial distriЬution of mtDNA lineages over the cemetery area under a simplified model that disregards the archaeological differentiation of the Andronovo time burials other than their spatial distribution. The analysis was performed at several resolution levels: from crude (at the level of the West Eurasian and East Eurasian clusters of mtDNA haplogroups) to high-resolution (at the level of specific structural variants). The information potential of each of those levels is evaluated. Data on the pattern of individual mtDNA lineage distribution are analyzed with regard to clusters on the cemetery area, indicating a considerable role of kinship across the site. The most promising areas for revealing groups of close relatives are identified. The findings enaЬle us to minimize the effect of close kinship on assessing the population structure of Tartas-1.

A 1.1 Mb duplication CNV on chromosome 17 contributes to skeletal muscle development in Boer goats.

The Boer goat is one of the top meat breeds in modern animal husbandry and has attracted widespread attention for its unique growth performance. However, the genetic basis of muscle development in the Boer goat remains obscure. In this study, we identified specific structural variants in the Boer goat based on genome-wide selection signals and analyzed the basis of the molecular heredity of related candidate genes in muscle development. A total of 9 959 autosomal copy number variations (CNVs) were identified through selection signal analysis in 127 goat genomes. Specifically, we confirmed that the highest signal CNV (HSV) was a chromosomal arrangement containing an approximately 1.11 Mb (CHIR17: 60062304-61171840 bp) duplicated fragment inserted in reverse orientation and a 5 362 bp deleted region (CHIR17:60145940-60151302 bp) with overlapping genes (e.g., ARHGAP10, NR3C2, EDNRA, PRMT9, and TMEM184C). The homozygous duplicated HSV genotype (+/+) was found in 96% of Boer goats but was not detected in Eurasian goats and was only detected in 4% of indigenous African goats. The expression network of three candidate genes ( ARHGAP10, NR3C2, and EDNRA) regulating dose transcription was constructed by RNA sequencing. Results indicated that these genes were involved in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs) and their overexpression significantly increased the expression of SAA3. The HSV of the Boer goat contributed to superior skeletal muscle growth via the dose effects of overlapping genes.

Genome structural variation in Escherichia coli O157:H7.

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.

Vulcan: Improved long-read mapping and structural variant calling via dual-mode alignment.

BackgroundLong-read sequencing has enabled unprecedented surveys of structural variation across the entire human genome. To maximize the potential of long-read sequencing in this context, novel mapping methods have emerged that have primarily focused on either speed or accuracy. Various heuristics and scoring schemas have been implemented in widely used read mappers (minimap2 and NGMLR) to optimize for speed or accuracy, which have variable performance across different genomic regions and for specific structural variants. Our hypothesis is that constraining read mapping to the use of a single gap penalty across distinct mutational hot spots reduces read alignment accuracy and impedes structural variant detection.FindingsWe tested our hypothesis by implementing a read-mapping pipeline called Vulcan that uses two distinct gap penalty modes, which we refer to as dual-mode alignment. The high-level idea is that Vulcan leverages the computed normalized edit distance of the mapped reads via minimap2 to identify poorly aligned reads and realigns them using the more accurate yet computationally more expensive long-read mapper (NGMLR). In support of our hypothesis, we show that Vulcan improves the alignments for Oxford Nanopore Technology long reads for both simulated and real datasets. These improvements, in turn, lead to improved accuracy for structural variant calling performance on human genome datasets compared to either of the read-mapping methods alone.ConclusionsVulcan is the first long-read mapping framework that combines two distinct gap penalty modes for improved structural variant recall and precision. Vulcan is open-source and available under the MIT License at https://gitlab.com/treangenlab/vulcan.

Gossypium Genomics: Trends, Scope, and Utilization for Cotton Improvement.

Cotton (Gossypium spp.) is the most important natural fiber crop worldwide. The diversity of Gossypium species also provides an ideal model for investigating evolution and domestication of polyploids. However, the huge and complex cotton genome hinders genomic research. Technical advances in high-throughput sequencing and bioinformatics analysis have now largely overcome these obstacles, bringing about a new era of cotton genomics. Here, we review recent progress in Gossypium genomics based on whole genome sequencing, resequencing, and comparative genomics, which have provided insights about the genomic basis of fiber biogenesis and the landscape of cotton functional genomics. We address current challenges and present multidisciplinary genomics-enabled breeding strategies covering the breadth of high fiber yield, quality, and environmental resilience for future cotton breeding programs.

Combining accurate tumor genome simulation with crowdsourcing to benchmark somatic structural variant detection

BackgroundThe phenotypes of cancer cells are driven in part by somatic structural variants. Structural variants can initiate tumors, enhance their aggressiveness, and provide unique therapeutic opportunities. Whole-genome sequencing of tumors can allow exhaustive identification of the specific structural variants present in an individual cancer, facilitating both clinical diagnostics and the discovery of novel mutagenic mechanisms. A plethora of somatic structural variant detection algorithms have been created to enable these discoveries; however, there are no systematic benchmarks of them. Rigorous performance evaluation of somatic structural variant detection methods has been challenged by the lack of gold standards, extensive resource requirements, and difficulties arising from the need to share personal genomic information.ResultsTo facilitate structural variant detection algorithm evaluations, we create a robust simulation framework for somatic structural variants by extending the BAMSurgeon algorithm. We then organize and enable a crowdsourced benchmarking within the ICGC-TCGA DREAM Somatic Mutation Calling Challenge (SMC-DNA). We report here the results of structural variant benchmarking on three different tumors, comprising 204 submissions from 15 teams. In addition to ranking methods, we identify characteristic error profiles of individual algorithms and general trends across them. Surprisingly, we find that ensembles of analysis pipelines do not always outperform the best individual method, indicating a need for new ways to aggregate somatic structural variant detection approaches.ConclusionsThe synthetic tumors and somatic structural variant detection leaderboards remain available as a community benchmarking resource, and BAMSurgeon is available at https://github.com/adamewing/bamsurgeon.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii, isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions.

Breaking the bilayer: OMV formation during environmental transitions.

Gram-negative bacteria maintain the barrier properties of the outer membrane (OM) in a wide array of physiological conditions despite their inability to degrade lipopolysaccharide (LPS) and protein material present in the outer leaflet of the OM. Through characterization of the native dynamics of outer membrane LPS change we recently described a mechanism in which these diderm organisms overcome this design flaw. In response to different environmental stimuli Salmonella enterica modulates the export of specific structural variants of lipid A via outer membrane vesicles (OMVs). We proposed that the polymorphic model for regulation of membrane lipid content could largely account for the structural differences between secreted and retained lipid A species. However, differences in OMV production levels and size observed between environmental conditions remain unexplained. Further exploration into the relationship between OMV production level and content specificity may shed light onto the enigmatic mechanisms of OMV formation.

Development of an analytical method for the unambiguous structure elucidation of cyclic peptides with special appliance for hepatotoxic desmethylated microcystins

The periodical occurrence of harmful algal blooms (HABs) in freshwater lakes requires the determination of potential cyanobacterial toxins, especially microcystins (MCs). On demand of an adequate risk assessment, the high diversity of these hepatotoxic cyclic heptapeptides implicates the need of an unambiguous detection of their specific structural variants. Therefore, LC–MS and LC–MS/MS methods are the approaches of choice for determination of MCs. In contrast, even tandem mass spectromic fragmentation patterns are not even sufficient in any kind of structural determination requirements, whereas NMR methods require very high amounts of MCs. In this study, we present a novel method for chromatographic separation of desmethylated microcystins (dm-MCs). Based on the isolation of the specific structural variants using semi-preparative HPLC, a method was developed for the structure elucidation of cyclic peptides with special appliance for the determination of dm-MCs via analysis of the specific amino acid composition after peptide hydrolysis followed by stereospecific detection of the amino acids and resulting keto acids. On the basis of this method it is demonstrated that dm-MC-RR with the structure [Dha 7]MC-RR represented the major compound in the microcystin pattern of Microcystis aeruginosa bloom events in 2005 and 2006 in Lake Senftenberg, Germany.

An investigation of the Al–Pd–Fe phase diagram between 50 and 100 at.% Al: phase equilibria at 750 °C

The isothermal 750 °C section of Al–Pd–Fe was determined between 50 and 100 at.% Al. The range of the Al–Pd orthorhombic ε-phases extends up to ∼10 at.% Fe. Apart from the four specific structural variants ε 6, ε 16, ε 22 and ε 28, irregular structures were observed in this continuous range. In addition to the ternary cubic C 1, C 2 phases and orthorhombic O-Al 13(Fe,Pd) 4 observed at higher temperatures, a new stable orthorhombic N-phase was revealed. It exhibits a structural relation to the ε-phases and decagonal quasicrystals.

Ultra-high resolution electron microscopy investigation of growth defects in CVD diamond films: twin interactions and fivefold twin centres

Abstract An elucidation of the core structure of fivefold twin centres arising from the interaction between less than five first-order twin boundaries in plasma-assisted chemical vapour deposition (CVD) of diamond films is reported. These particular twinning centres have been identified by ultra-high resolution electron microscopy at 0.12 nm resolution, with the help of image calculations. Plausible three-dimensional atomic-scale models are proposed for two specific structural variants, which have been found closely connected to high-order twin boundaries via original heptagonal or octagonal structural units. To our knowledge, this is the first time that such types of fivefold twin centres and associated structural units, which are quite representative of the growth defects observed in CVD diamond, have been reported.