Complex Structural Variants Research Articles

Optical Genome Mapping as a Tool to Unveil New Molecular Findings in Hematological Patients with Complex Chromosomal Rearrangements.

Standard cytogenetic techniques (chromosomal banding analysis-CBA, and fluorescence in situ hybridization-FISH) show limits in characterizing complex chromosomal rearrangements and structural variants arising from two or more chromosomal breaks. In this study, we applied optical genome mapping (OGM) to fully characterize two cases of complex chromosomal rearrangements at high resolution. In case 1, an acute myeloid leukemia (AML) patient showing chromothripsis, OGM analysis was fully concordant with classic cytogenetic techniques and helped to better refine chromosomal breakpoints. The OGM results of case 2, a patient with non-Hodgkin lymphoma, were only partially in agreement with previous cytogenetic analyses and helped to better define clonal heterogeneity, overcoming the bias related to clonal selection due to cell culture of cytogenetic techniques. In both cases, OGM analysis led to the identification of molecular markers, helping to define the pathogenesis, classification, and prognosis of the analyzed patients. Despite extensive efforts to study hematologic diseases, standard cytogenetic methods display unsurmountable limits, while OGM is a tool that has the power to overcome these limitations and provide a cytogenetic analysis at higher resolution. As OGM also shows limits in defining regions of a repetitive nature, combining OGM with CBA to obtain a complete cytogenetic characterization would be desirable.

Haplotype-resolved assemblies and variant benchmark of a Chinese Quartet

BackgroundRecent state-of-the-art sequencing technologies enable the investigation of challenging regions in the human genome and expand the scope of variant benchmarking datasets. Herein, we sequence a Chinese Quartet, comprising two monozygotic twin daughters and their biological parents, using four short and long sequencing platforms (Illumina, BGI, PacBio, and Oxford Nanopore Technology).ResultsThe long reads from the monozygotic twin daughters are phased into paternal and maternal haplotypes using the parent–child genetic map and for each haplotype. We also use long reads to generate haplotype-resolved whole-genome assemblies with completeness and continuity exceeding that of GRCh38. Using this Quartet, we comprehensively catalogue the human variant landscape, generating a dataset of 3,962,453 SNVs, 886,648 indels (< 50 bp), 9726 large deletions (≥ 50 bp), 15,600 large insertions (≥ 50 bp), 40 inversions, 31 complex structural variants, and 68 de novo mutations which are shared between the monozygotic twin daughters. Variants underrepresented in previous benchmarks owing to their complexity—including those located at long repeat regions, complex structural variants, and de novo mutations—are systematically examined in this study.ConclusionsIn summary, this study provides high-quality haplotype-resolved assemblies and a comprehensive set of benchmarking resources for two Chinese monozygotic twin samples which, relative to existing benchmarks, offers expanded genomic coverage and insight into complex variant categories.

CloveBiotech: Complex structural variant calling for engineered microbial strains

Abstract This paper introduces CloveBiotech, an extension and optimization of CLOVE, which is designed to accurately classify complex structural variants (SVs) resulting from genetic modifications in the genomes of microbial strains. While CLOVE covers various complex SV types, it lacks support for large insertions, targeted vector integrations and substitutions of genes with a marker cassette. CloveBiotech addresses these limitations by modifying the algorithm to handle more diverse SV scenarios, including new fusion patterns. However, covering these scenarios demanded a major rewrite of multiple classes and functions. Performance evaluations on simulated data demonstrate its improved precision and recall rates of up to 0.92 and 0.96 respectively, across different sequencing parameters. With these enhancemanets, CloveBiotech provides a valuable tool for scientists working with genetically modified microorganisms, facilitating accurate SV detection and classification.

Whole-genome long-read sequencing downsampling and its effect on variant-calling precision and recall.

Advances in long-read sequencing (LRS) technologies continue to make whole-genome sequencing more complete, affordable, and accurate. LRS provides significant advantages over short-read sequencing approaches, including phased de novo genome assembly, access to previously excluded genomic regions, and discovery of more complex structural variants (SVs) associated with disease. Limitations remain with respect to cost, scalability, and platform-dependent read accuracy and the tradeoffs between sequence coverage and sensitivity of variant discovery are important experimental considerations for the application of LRS. We compare the genetic variant-calling precision and recall of Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) HiFi platforms over a range of sequence coverages. For read-based applications, LRS sensitivity begins to plateau around 12-fold coverage with a majority of variants called with reasonable accuracy (F1 score above 0.5), and both platforms perform well for SV detection. Genome assembly increases variant-calling precision and recall of SVs and indels in HiFi data sets with HiFi outperforming ONT in quality as measured by the F1 score of assembly-based variant call sets. While both technologies continue to evolve, our work offers guidance to design cost-effective experimental strategies that do not compromise on discovering novel biology.

Single Cell Whole Genome Measurement of Genomic Heterogeneity and Clonal Evolution in Multiple Myeloma

Background: Sequencing studies have advanced our understanding of the genomic underpinnings of Multiple Myeloma (MM). While whole genome sequencing (WGS) has emerged as a comprehensive approach for profiling the genomic landscape of MM, WGS has limited sensitivity in defining the intrapatient genomic heterogeneity. Single cell RNA approaches can capture phenotypic heterogeneity but fail to recover most of the key genomic drivers. To overcome these historical limitations, we utilized a newer single cell whole genome direct library preparation (scWGS DLP+; Lacks et al. Cell 2019) sequencing technology able to detect early changes and study clonal population dynamics, providing a more precise understanding of the emergence and clinical impact of genomic events during MM evolution. Methods: scWGS DLP+ was applied to CD138 + Plasma Cells (PC) isolated from bone marrow samples of MM patients. Using newly developed computational approaches, we characterized the Single Nucleotide Variants (SNVs), copy number changes and complex Structural Variants (SVs) at a single cell level and use this information to reconstruct the evolutionary histories of each patient's myeloma. Results: We applied DLP to 7 MM patients obtaining 800-1600 single cell whole genomes per patient. All canonical IgH translocations identified by FISH were recovered in the scWGS data. Single cell copy number heterogeneity was observed in all but 1 patient, including heterogeneity affecting known drivers. The number of subclones ranged from 1 to 15, with an average of 6 subclones detected per patient (≥10 cells, ≥5MB distinct segmental difference). This is in striking contrast with bulk WGS where we are usually able to define no more than one or two subclones. Gain of 1q (3 copies) was observed in all patients but was either rare (1-3%, 3 patients) or clonal (&amp;gt;99%, 4 patients), with two clonal 1q patients exhibiting convergent subclonal 1q amplification (&amp;gt;3 copies). Loss of previously gained chromosomes was observed including loss of ancestrally gained chromosome 15 in hypodiploid Patient 2, and sporadic loss of 1q in patients with ubiquitous 1q gain. For 3 of 7 patients, hypodiploidy (HRD) had arisen before the most recent clonal expansion. HRD clones continued to diversify, accruing multiple additional events on HRD chromosomes including large or focal gains, or deletions subsequent to a gain. Interestingly, one case had 1 small hypodiploid clone co-existing with a completely different major clone with 1q and t(14;20) IgH translocation, suggesting the existence of two distinct clonal plasma cell populations (one malignant and one likely bening). Two of the 7 patients harbored both small (&amp;lt;20 cells) whole genome doubled (WGD) subclones, and small numbers of highly aneuploid likely produced by multi-polar cell division post WGD. We explored two patient's evolutionary histories in detail, highlighting the unique resolution of scWGS DLP+ to detect CNV but also SNV, mutational signatures and SVs. Patient 3 exhibited 4 distinct subclones with distant evolutionary origin as evidenced by the relatively small number of shared SNV. APOBEC signature mutations were detected in all 4 subclones suggesting APOBEC mutagenesis was still ongoing across the cell populations in this patient. In Patient 1, a complex SV on chromosome 17 resulted in loss of most of 17q and an amplification of 17q segments. The heterogeneous structure of chromosome 17 across clones suggests a late onset of this complex event and ongoing instability. Chromosome 16 exhibited a pattern of cell specific arm loss likely resulting from telomere loss. Interestingly, a minor clone showed complex amplification of 16p affecting TNFRSF17, suggesting that chromosome 16 is at an earlier stage of a process of instability that starts with cell specific arm loss, and ends with clonal expansion of a complex amplification SV targeting a gene with fitness benefit. Discussion: Applied to Multiple Myeloma, scWGS shows higher clonal heterogeneity than previously described with multiple clones exhibiting diverse genetic alterations. The findings underscore the genomic complexity and dynamic nature of MM, highlighting the need for sensitive techniques like scWGS to capture the full spectrum and chronological order of genomic events.

The Complex Structural Variant Chromothripsis Can be Defined on Targeted Sequencing Panels, Allowing Direct Clinical Translation in Order to Improve Multiple Myeloma Prognostication

Introduction: the complex structural variant (SV) chromothripsis is an independent predictor of progression-free (PFS) and overall survival (OS) in newly diagnosed multiple myeloma (NDMM); predictive in multivariate analysis when considering ISS, IgH translocations and TP53 status (Rustad BCD 2020). Chromothripsis is not included in current prognostic scores, despite a prevalence of 24% in NDMM. While gold-standard definition requires whole genome sequencing (WGS), we've previously shown that copy-number (CN) signatures are also able to accurately define chromothripsis in whole exome sequencing (WES, Maclachlan Nat Com 2021). As neither WGS nor WES are currently used in clinical medicine, we applied CN signatures to targeted sequencing, aiming for direct clinical translation of this key biological feature into clinical practice. Memorial Sloan Kettering (MSK) now offers the MSK-Heme-IMPACT targeted sequencing panel to all MM patients having a bone marrow biopsy (BMBx), as well as offering retrospective analysis for current patients having leftover DNA from prior BMBx. Methods: We analyzed 420 samples having either myTYPE or MSK-Heme-IMPACT targeted sequencing at MSK prior to July 2023. We excluded those with whole genome duplication, applied CN signatures, then for samples from NDMM performed multivariate analysis for PFS prediction by backwards stepwise regression. In samples with CN signatures predictive for chromothripsis and with DNA available from the same BMBx, we performed parallel WGS in order to check for chromothripsis. WGS sequencing was at 60-80x for tumor, 30-40x for normal, with analysis including Battenberg for CN analysis, with SvABA, BRASS and GRIDSS for SV calling. Additional parallel WGS is in progress for 45 samples with NDMM prior to DVRd or DKRd, in order to examine chromothripsis prediction and PFS impact in the context of highly effective induction. Results: From 420 samples, 48 were collected at the precursor stage, 205 with NDMM, and 167 with relapsed disease. 39 patients had 2 assays performed, while the remainder were unique patient samples. Within NDMM, the range of induction regimens received included lenalidomide and dexamethasone with bortezomib (VRd), carfilzomib (KRd) or daratumumab (DRd), along with quadruplet therapy (DVRd or DKRd). Targeted sequencing data detected all 6 key CN features observed in WGS and WES analysis. Similar to WES, there was a lower contribution from very high breakpoint counts per 10mB and longer lengths of oscillating CN segments, due to the lower resolution of targeted sequencing data. De novo extraction of CN signatures defined 1 signature containing multiple features consistent with chromothripsis; high breakpoint count per 10mB, more jumps between adjacent CN segments, longer lengths of oscillating CN segments and a predominance of small CN segments. When examining samples from NDMM, contribution from this signature was predictive of PFS in multivariate analysis when considering age, ISS, t(4;14), TP53 status and gain1q21 (hazard ratio &amp;gt; 4, p=0.003). 33 samples had a predictive probability for chromothripsis &amp;gt; 0.6 (the cut-off in WGS / WES having the highest sensitivity and specificity for prediction), with also having further DNA available. 30/33 (91%) were confirmed to harbor chromothripsis on manual inspection of WGS data. 19/30 (63%) with chromothripsis were BMBx from NDMM, while 11/30 (37%) were from relapsed disease. Discussion: the complex SV chromothripsis is a key genomic risk factor predicting for poor PFS and OS in MM, which is currently not being captured in routine practice or in clinical trials. Here, we demonstrate that chromothripsis can be predicted from targeted sequencing data, confirmed by gold-standard WGS analysis. Ongoing studies are expanding our cohort of NDMM with concurrent targeting sequencing and WGS, to define the sensitivity and specificity of targeted-sequencing-based CN signature prediction of chromothripsis. The additional cohort largely comprises patients treated with daratumumab-quadruplet induction therapy, aiming to confirm that chromothripsis and CN signatures retain prognostic significance in the context of this therapy. With this data is in hand, we will develop a CN signature fitting tool, similar to mmsig for mutational signatures (Rustad Comm Biol 2021), to allow easily accessible incorporation of chromothripsis assessment into clinical practice.

Comprehensive Genomic Characterization of Response and Resistance to Daratumumab-Based Quadruplet Induction in Newly Diagnosed Multiple Myeloma Patients

Introduction: Current prognostic scores for newly diagnosed multiple myeloma (NDMM) rely on measures of tumor bulk and limited genomic information. A recent artificial-intelligence-based model suggested that individualized prognostication and prediction of response to specific therapies may be improved by more comprehensive genomic assessment (IRMMa, Maura et al Blood 2022). The 20 significant genomic contributors included APOBEC mutational activity, gain/amp1q, TP53 aberration, and loss of any of CDKN2C, RPL5, FUBP1, RB1, RASA2, 10q23 or 12p13. The highest impact on progression-free (PFS) and overall survival (OS) came from copy number (CN) signature profiles suggesting the structural variant chromothripsis (Maclachlan et al Nat Comm 2021). We have previously demonstrated that CN features consistent with chromothripsis can be detected in targeted sequencing data, potentially increasing clinical application (Maclachlan el at Blood 2020). One limitation of the recently presented IRMMa model was an absence of patients prescribed daratumumab-based quadruplet induction, (dara-quads; daratumumab, lenalidomide, dexamethasone with carfilzomib [DKRd] or bortezomib [DVRd]). Methods: We applied comprehensive genomic assessment to a cohort of 234 NDMM patients from Memorial Sloan Kettering Cancer Center initiated on dara-quad induction prior to May 2023. Genomic data included FISH (n=215), SNP-array (n=127), targeted sequencing (MSK-IMPACT-Heme, n=78) and whole genome sequencing (WGS, n=45). A further 80 samples are undergoing MSK-IMPACT-Heme (n=25) and WGS (n=55) currently. Results: Among 234 patients analyzed, 86 patients received DKRd and 148 DVRd. Overall median follow-up was 1.4 years (y); 4.2y with DKRd (IQR 1.9-4.7, max 5.7) and 1y with DVRd (IQR 0.5-1.4, max 3.4). 25 patients progressed following DKRd and 20 following DVRd, with 9 progressing during induction chemotherapy. ISS stage did not predict for PFS or OS, either in the whole cohort, or in individual regimens. The inclusion of genomic assessment improved prognostication; while R-ISS, high-risk cytogenetics as per R-ISS, and R2-ISS each improved PFS prediction (p&amp;lt;0.01), consideration of every feature assessable by FISH (including del1p, amp1q, t(14;20), MYC-translocations) further increased discrimination (p=0.009). Assessing as per the Master study (Costa JCO 2022), 2+ co-occurring high-risk features predicted for shorter PFS when compared with 1 or 0 risk factors (p=0.002). Within the 9 who progressed during induction, 4 were ISS III, 6 had a risk factor evident via FISH, and 4 had multiple high-risk features, while 4 had no risk factors by standard methods. Using comprehensive genomic assessment (SNP-array, MSK-IMPACT-Heme and WGS) allowed for the assessment of additional genomic features in those with adequate follow up (predominantly DKRd). Del1p22.1 ( RPL5) was strongly predictive of shorter PFS (p&amp;lt;0.0001), as were del22q12.1 ( XBP1, p=0.02) and high APOBEC-mutational activity (p=0.01). Additional features demonstrated a trend to significance, including del1p12 ( TENT5C, p=0.06) and the complex structural variant chromothripsis. Parallel performance of MSK-IMPACT-Heme and WGS on the same samples allowed demonstration that CN signatures detected by targeted sequencing were accurate indications of complex chromothripsis events. The expanded WGS cohort will further define sensitivity and specificity of this detection. Discussion: In the context of highly effective dara-quad induction, ISS stage did not predict for either PFS or OS. Limited cytogenetic assessment improved prognostication, while advanced genomic assessment was highly discriminatory. The current study provides the rationale for more comprehensive genomic assessment in patients receiving dara-quads in order to refine prognostication and consider novel treatment approaches for high-risk patients. We are significantly expanding our cohort of WGS in dara-quad-treated patients, formalizing the accuracy for CN signature detection of chromothripsis from targeted sequencing data, allowing application in routine clinical practice.

Unraveling Diverse Mechanisms of Complex Structural Variant Interactions through Multiomic Data in Multiple Myeloma

Introduction: Structural variants (SVs) play a role in proliferation of plasma cells that characterizes multiple myeloma (MM). Several hallmark SVs have been identified in MM, including translocations involving the immunoglobulin heavy chain locus and several partner genes, as well as deletions and duplications affecting key genes such as TP53, RB1, and MYC. Complex SVs such as templated insertions, chromothripsis, and chromoplexy are associated with disease progression but have not yet systematically been studied. Methods: Paired short-read (Illumina, average depth 75x), long-read HiFi (PacBio, 16x), optical genome mapping (OGM; Bionano Genomics, 341x) and Micro-C (Cantata Bio, 45x) data were sequenced from 8 relapsed MM patient-derived xenograft samples and 2 cell lines, covering t(11;14), t(4;14), and hyperdiploid subgroups. Reads were aligned to the hg38 reference genome while complex SVs (chromothripsis, chromoplexy and templated insertion) were systematically identified and accurately reconstructed by combining short-read (Manta), HiFi (pbsv), OGM, and Micro-C data (EagleC). Chromosome organization including loops, topologically associated domains (TADs) and A/B compartments were called from Micro-C data (Mustache and Juicer). Haplotype phasing was conducted using small variants detected from HiFi data. De novo assembly of the genome was conducted using HiFi and Micro-C data and compared to the reference genome. Reconstructed derivative chromosome interactions due to complex SVs were further identified by aligning Micro-C reads to the newly assembled genomes. Previously identified super-enhancers from H3K27Ac ChIP-seq from MM cell lines were mapped onto these SVs and expression of target genes assessed by RNA-seq. Results: 1,340 SVs were found in 10 samples using OGM of which 828 were identified in short-read WGS data. 16 complex SVs including chromothripsis, chromoplexy and templated insertions were detected in 10 samples, and we describe two (Fig.1) in detail here. A complex templated insertion consisting of a t(2;6;8) was found in a t(11;14) PDX sample with copy number gains at breakpoints which could not be resolved from short-read data alone. OGM data revealed a single contig consisting of DNA from chromosomes 2, 6, and 8. Micro-C data revealed interactions between these chromosomes, specifically the gained regions on chromosomes 2 and 8 showed more interactions with chromosome 6 than with other chromosomes, indicating insertions into chromosome 6. As a result, both MYC at chromosome 8 and GALM at chromosome 2 were inserted into the TXNDC5 locus, containing a known MM super-enhancer, leading to the over-expression of both MYC and GALM (log 2FC=1.7 and 1.0) in this sample. A de novo genome assembly indicated neo-TAD formation encompassing the super-enhancer and both genes. In coMMpass dataset, high expression of GALM and MYC are significantly associated with inferior overall (p=0.002 and 0.05, logrank test) and progression-free survival (p=0.01 and 0.09). Another complex templated insertion consisting of a t(3;4;16) was identified in a hyperdiploid PDX sample, again with copy number gains at breakpoints. Although the short-read data indicated an assembly similar to the previous templated insertion SV, the OGM and Micro-C data indicated a 3-way chromosome swap where the short arm of chromosome 3 was translocated to the short arm of chromosome 4, the short arm of chromosome 4 was translocated to the long arm of chromosome 16, and the long arm of chromosome 16 was translocated to the short arm of chromosome 3. Separate contigs for the t(4;16), t(3;16) and t(3;4) were confirmed by OGM data and only 2-way chromosomal interactions were seen in the Micro-C data. This complex event led to numerous oncogenes juxtaposed to super-enhancers, which caused over-expression of target genes such as ACD and RANBP10 (log 2FC=1.0 and 0.4), both of which were on chromosome 16 and are significantly associated with inferior overall (p=0.02 and p=0.004) and progression-free survival (p=0.003 and p=0.005). Conclusion: By utilizing multiomic techniques we were able to reconstruct complex SV events, showing different mechanisms of generating templated insertion events and identifying their biological consequences. We demonstrated how these complex events could potentially facilitate tumor proliferation via engaging the super-enhancers and regulating the expression of key oncogenes.

Whole-Genome Sequencing Identifies Structural Variation As a Key Driver of Disease Relapse and Aggressive Clinical Behavior in Multiple Myeloma

INTRODUCTION: Many key genomic events leading to relapse in multiple myeloma (MM) occur outside of coding regions and can only be identified using whole-genome sequencing (WGS). Studies of MM progression examining single nucleotide variants (SNVs) and insertions/deletions (InDels) have identified a distinct pattern of mutations associated with resistance, however these do not fully account for drivers of progression. Studies of relapsed patients provide hints about the molecular mechanisms driving progression based on identification of homozygous loss of tumor suppressor genes, aberrant copy-number variants (CNVs), and rare examples of complex structural variants (SV). We have analyzed a set of presentation and relapsed MM samples, including paired data, derived from WGS of plasma cells and focused on SVs as key drivers of progression. METHODS: WGS was performed on 338 MM patient samples using CD138-sorted plasma cells, among which 237 samples were paired including 101 patients (30%) at presentation and at relapse, 35 of whom had more than one sample at relapse. A bioinformatics pipeline employing a consensus mechanism for determining the final set of somatic events was used based on Mutect2, Strelka2, and VarScan2 for SNVs; Mutect2, Strelka2, VarScan2, and SvABA for InDels; Battenberg and FACETS for CNVs; Manta, SvABA, DELLY2, and IgCaller for SVs. Additionally, an admixture workflow was used to estimate each individual's ancestral lineage using continentally-distinct references, comprising 23 regional populations within 5 super-populations from the 1000 Genomes Project (https://github.com/pblaney/mgp1000). Complex rearranged genomes were reconstructed using the graph-based R package JaBbA (https://github.com/mskilab-org/JaBbA). Additionally, the python package Pairtree (https://github.com/morrislab/pairtree) was used to describe the evolutionary history of acquired mutations in these patients. RESULTS: Themedian age of the cases studied was 67, and 42% were female. Racial admixture correlated with self-identification, including 74% of European ancestry with the remainder being predominantly of African ancestry. High-risk cytogenetic features were found in 10% and 13% at diagnosis and relapse, respectively. As expected, the tumor mutational burden of the relapse cases was higher than that at presentation. The patterns of SNV mutational drivers were similar at relapse in comparison to presentation with again evidence for a role of biallelic tumor suppressor gene inactivation as being a key mechanism. We also found that the number of SVs increased at relapse. Complex SVs including templated insertions, chromoplexy, and chromothripsis were detected and occurred exclusively at relapse in some cases as well as earlier in the natural history in others, suggesting they can occur as both early and late driver events. Characterizing complex SVs, further we identified them at lower levels in cases at presentation compared to relapse, consistent with these being at a higher clonal fraction and providing further evidence for their role as driver events. We did not see the emergence of additional rearrangements at relapse in either the templated insertions or chromoplexy events, suggesting that these occur at a single timepoint and remain structurally stable overtime. CONCLUSIONS: Complex SVs provide a novel mechanism driving relapse in MM which can deregulate multiple genes simultaneously providing new potential markers of aggressive disease behavior and disease evolution.

A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants

BackgroundThe red junglefowl, the wild outgroup of domestic chickens, has historically served as a reference for genomic studies of domestic chickens. These studies have provided insight into the etiology of traits of commercial importance. However, the use of a single reference genome does not capture diversity present among modern breeds, many of which have accumulated molecular changes due to drift and selection. While reference-based resequencing is well-suited to cataloging simple variants such as single-nucleotide changes and short insertions and deletions, it is mostly inadequate to discover more complex structural variation in the genome.MethodsWe present a pangenome for the domestic chicken consisting of thirty assemblies of chickens from different breeds and research lines.ResultsWe demonstrate how this pangenome can be used to catalog structural variants present in modern breeds and untangle complex nested variation. We show that alignment of short reads from 100 diverse wild and domestic chickens to this pangenome reduces reference bias by 38%, which affects downstream genotyping results. This approach also allows for the accurate genotyping of a large and complex pair of structural variants at the K feathering locus using short reads, which would not be possible using a linear reference.ConclusionsWe expect that this new paradigm of genomic reference will allow better pinpointing of exact mutations responsible for specific phenotypes, which will in turn be necessary for breeding chickens that meet new sustainability criteria and are resilient to quickly evolving pathogen threats.

Long-Read Sequencing Resolves a Complex Structural Variant in PRKN Parkinson's Disease.

Parkin RBR E3 ubiquitin-protein ligase (PRKN) mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). PRKN is located in FRA6E, which is one of the common fragile sites in the human genome, making this region prone to structural variants. However, complex structural variants such as inversions of PRKN are seldom reported, suggesting that there are potentially unrevealed complex pathogenic PRKN structural variants. To identify complex structural variants in PRKN using long-read sequencing. We investigated the genetic cause of monozygotic twins presenting with a young onset dystonia-parkinsonism using targeted sequencing, whole exome sequencing, multiple ligation probe amplification, and long-read sequencing. We assessed the presence and frequency of complex inversions overlapping PRKN using whole-genome sequencing data of Accelerating Medicines Partnership Parkinson's disease (AMP-PD) and United Kingdom (UK)-Biobank datasets. Multiple ligation probe amplification identified a heterozygous exon three deletion in PRKN and long-read sequencing identified a large novel inversion spanning over 7 Mb, including a large part of the coding DNA sequence of PRKN. We could diagnose the affected subjects as compound heterozygous carriers of PRKN. We analyzed whole genome sequencing data of 43,538 participants of the UK-Biobank and 4941 participants of the AMP-PD datasets. Nine inversions in the UK-Biobank and two in AMP PD were identified and were considered potentially damaging and likely to affect PRKN expression. This is the first report describing a large 7 Mb inversion involving breakpoints outside of PRKN. This study highlights the importance of using long-read sequencing for structural variant analysis in unresolved young-onset PD cases. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Unexpected structural complexity of d-block metallosupramolecular architectures within the benzimidazole-phenoxo ligand scaffold for crystal engineering aspects

Design of metallosupramolecular materials encompassing more than one kind of supramolecular interaction can become deceptive, but it is necessary to better understand the concept of the controlled formation of supramolecular systems. Herein, we show the structural diversity of the bis-compartmental phenoxo-benzimidazole ligand H3L1 upon self-assembly with variety of d-block metal ions, accounting for factors such as: counterions, pH, solvent and reaction conditions. Solid-state and solution studies show that the parent ligand can accommodate different forms, related to (de)protonation and proton-transfer, resulting in the formation of mono-, bi- or tetrametallic architectures, which was also confirmed with control studies on the new mono-compartmental phenoxo-benzimidazole H2L2 ligand analogue. For the chosen architectures, structural variables such as porous character, magnetic behaviour or luminescence studies were studied to demonstrate how the form of H3L1 ligand affects the final form of the supramolecular architecture and observed properties. Such complex structural variations within the benzimidazole-phenoxo-type ligand have been demonstrated for the first time and this proof-of-concept can be used to integrate these principles in more sophisticated architectures in the future, combining both the benzimidazole and phenoxide subunits. Ultimately, those principles could be utilized for targeted manipulation of properties through molecular tectonics and crystal engineering aspects.

Clinical applications of next-generation sequencing in the diagnosis of genetic disorders in Korea: a narrative review

Background: Next-generation sequencing (NGS) technologies have revolutionized genetic testing and enabled efficient screening of various genetic conditions in clinical settings. However, the clinical application of genetic test results presents numerous significant challenges. This review aims to provide a comprehensive overview of key concepts for the clinical application of NGS, including (1) technical aspects and limitations, (2) variant classification, (3) clinical interpretation, (4) familial testing and genetic counseling, and (5) ethical considerations.Current Concepts: In short-read-based NGS, several limitations exist in detecting genomic variations, including repetitive sequences or complex structural variations. The variant classification process can be influenced by suspected genomic conditions and the accessibility of genomic databases. Therefore, the final genetic diagnosis depends on the physician’s discretion, which relies on the genotype-phenotype correlations and reverse phenotyping through additional evaluations. Familial testing can help trace the origin of variants and allele segregation and aid in variant interpretation, risk assessment, disease prevention, and family planning. In addition to addressing the clinical impact of genetic results, genetic counselors should also consider potential consequences related to ethical, legal, and social issues, including family dynamics.Discussion and Conclusion: NGS-based genetic testing is a promising diagnostic tool for genetic disorders, but proper variant interpretation and clinical evaluation are crucial for optimal clinical practice. Notably, ethical considerations and regulatory measures are required to prepare for the next era of genomic medicine.

OC 57.4 Deciphering Novel Complex Structural Variations in Severe Hemophilia a Patients by Optical Genome Mapping

OC 57.4 Deciphering Novel Complex Structural Variations in Severe Hemophilia a Patients by Optical Genome Mapping

SoyDNGP: a web-accessible deep learning framework for genomic prediction in soybean breeding.

Soybean is a globally significant crop, playing a vital role in human nutrition and agriculture. Its complex genetic structure and wide trait variation, however, pose challenges for breeders and researchers aiming to optimize its yield and quality. Addressing this biological complexity requires innovative and accurate tools for trait prediction. In response to this challenge, we have developed SoyDNGP, a deep learning-based model that offers significant advancements in the field of soybean trait prediction. Compared to existing methods, such as DeepGS and DNNGP, SoyDNGP boasts a distinct advantage due to its minimal increase in parameter volume and superior predictive accuracy. Through rigorous performance comparison, including prediction accuracy and model complexity, SoyDNGP represents improved performance to its counterparts. Furthermore, it effectively predicted complex traits with remarkable precision, demonstrating robust performance across different sample sizes and trait complexities. We also tested the versatility of SoyDNGP across multiple crop species, including cotton, maize, rice and tomato. Our results showed its consistent and comparable performance, emphasizing SoyDNGP's potential as a versatile tool for genomic prediction across a broad range of crops. To enhance its accessibility to users without extensive programming experience, we designed a user-friendly web server, available at http://xtlab.hzau.edu.cn/SoyDNGP. The server provides two features: 'Trait Lookup', offering users the ability to access pre-existing trait predictions for over 500 soybean accessions, and 'Trait Prediction', allowing for the upload of VCF files for trait estimation. By providing a high-performing, accessible tool for trait prediction, SoyDNGP opens up new possibilities in the quest for optimized soybean breeding.

PL02.2.A RAPID, COMPREHENSIVE REPORTING OF MOLECULAR DIAGNOSTICS WITH RAPID-CNS2: A PROSPECTIVE VALIDATION COHORT

Abstract BACKGROUND The WHO classification of CNS tumours 2021 recommends reporting a wide range of molecular alterations for WHO-compatible diagnoses. Conventional molecular diagnostic workflows warrant considerable investment and long turnaround times limited by batching. Nanopore sequencing enables long read sequencing, real-time targeting and simultaneous base modification detection with compact devices. Rapid-CNS2- a rapid, comprehensive adaptive sampling based sequencing pipeline with a turnaround time of 5 days for CNS tumours was previously described. This study presents an optimised Rapid-CNS2 pipeline and prospective validation cohort that successfully reports hard-to-detect variants. MATERIAL AND METHODS We performed targeted adaptive sampling-based sequencing on 156 samples comprising a mix of archival and current diagnostic cases from the Department of Neuropathology, University Hospital Heidelberg. We tested variable conditions including minimum DNA input, target size, flowcell reuse and sequencing times. An analysis pipeline for detection of single nucleotide variants (SNV), indels, copy number alterations (CNA), fusions, complex structural variants (SV), target gene methylation and methylation classification incorporating multi-GPU optimization was deployed. Results were compared to NGS panel sequencing and EPIC array analyses. RESULTS Integrated diagnoses for all cases were in accordance with those issued using molecular alterations reported by conventional analyses. Rapid-CNS2 showed improved resolution over NGS panel sequencing for CNAs. Complete concordance was achieved for MGMT promoter methylation status and methylation classification. High accuracy was obtained for detection of SNV/Indels. Long reads spanning the breakpoint identified clinically relevant fusions. Subclonal SVs including EGFR vIII missed by NGS panel sequencing were detected with high confidence. The optimized workflow shortened the turnaround time to 30 hours from sample receipt to report and minimum input DNA required was reduced to 500ng. Due to the versatility of adaptive sampling, the analyses can readily be limited to a smaller set of targets or methylation and CNAs only, further reducing the turnaround time. CONCLUSION We present a thorough validation and prospective clinical application of Rapid-CNS2 with cases spanning a range of brain tumours and metastases. The improved custom neurooncology adaptive sampling-based sequencing pipeline enables simultaneous copy-number, mutational, structural variant and methylation analysis with flexible target selection, no additional library preparation, competitive costs and next-day reporting of RESULTS

Extrachromosomal circular DNA and structural variants highlight genome instability in Arabidopsis epigenetic mutants

Abundant extrachromosomal circular DNA (eccDNA) is associated with transposable element (TE) activity. However, how the eccDNA compartment is controlled by epigenetic regulations and what is its impact on the genome is understudied. Here, using long reads, we sequence both the eccDNA compartment and the genome of Arabidopsis thaliana mutant plants affected in DNA methylation and post-transcriptional gene silencing. We detect a high load of TE-derived eccDNA with truncated and chimeric forms. On the genomic side, on top of truncated and full length TE neo-insertions, we detect complex structural variations (SVs) notably at a disease resistance cluster being a natural hotspot of SV. Finally, we serendipitously identify large tandem duplications in hypomethylated plants, suggesting that SVs could have been overlooked in epigenetic mutants. We propose that a high eccDNA load may alter DNA repair pathways leading to genome instability and the accumulation of SVs, at least in plants.

Resolving complex structural variants via nanopore sequencing.

The recent development of high-throughput sequencing platforms provided impressive insights into the field of human genetics and contributed to considering structural variants (SVs) as the hallmark of genome instability, leading to the establishment of several pathologic conditions, including neoplasia and neurodegenerative and cognitive disorders. While SV detection is addressed by next-generation sequencing (NGS) technologies, the introduction of more recent long-read sequencing technologies have already been proven to be invaluable in overcoming the inaccuracy and limitations of NGS technologies when applied to resolve wide and structurally complex SVs due to the short length (100-500bp) of the sequencing read utilized. Among the long-read sequencing technologies, Oxford Nanopore Technologies developed a sequencing platform based on a protein nanopore that allows the sequencing of "native" long DNA molecules of virtually unlimited length (typical range 1-100Kb). In this review, we focus on the bioinformatics methods that improve the identification and genotyping of known and novel SVs to investigate human pathological conditions, discussing the possibility of introducing nanopore sequencing technology into routine diagnostics.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project

BackgroundCurrent clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm...

Linked-read based analysis of the medulloblastoma genome.

Medulloblastoma is the most common type of malignant pediatric brain tumor with group 4 medulloblastomas (G4 MBs) accounting for 40% of cases. However, the molecular mechanisms that underlie this subgroup are still poorly understood. Point mutations are detected in a large number of genes at low incidence per gene while the detection of complex structural variants in recurrently affected genes typically requires the application of long-read technologies. Here, we applied linked-read sequencing, which combines the long-range genome information of long-read sequencing with the high base pair accuracy of short read sequencing and very low sample input requirements. We demonstrate the detection of complex structural variants and point mutations in these tumors, and, for the first time, the detection of extrachromosomal DNA (ecDNA) with linked-reads. We provide further evidence for the high heterogeneity of somatic mutations in G4 MBs and add new complex events associated with it. We detected several enhancer-hijacking events, an ecDNA containing the MYCN gene, and rare structural rearrangements, such a chromothripsis in a G4 medulloblastoma, chromoplexy involving 8 different chromosomes, a TERT gene rearrangement, and a PRDM6 duplication.