Pan-cancer whole-genome comparison of primary and metastatic solid tumours.

Metastatic cancer remains an almost inevitably lethal disease1-3. A better understanding of disease progression and response to therapies therefore remains of utmost importance. Here we characterize the genomic differences between early-stage untreated primary tumours and late-stage treated metastatic tumours using a harmonized pan-cancer analysis (or reanalysis) of two unpaired primary4 and metastatic5 cohorts of 7,108 whole-genome-sequenced tumours. Metastatic tumours in general have a lower intratumour heterogeneity and a conserved karyotype, displaying only a modest increase in mutations, although frequencies of structural variants are elevated overall. Furthermore, highly variable tumour-specific contributions of mutational footprints of endogenous (for example, SBS1 and APOBEC) and exogenous mutational processes (for example, platinum treatment) are present. The majority of cancer types had either moderate genomic differences (for example, lung adenocarcinoma) or highly consistent genomic portraits (for example, ovarian serous carcinoma) when comparing early-stage and late-stage disease. Breast, prostate, thyroid and kidney renal clear cell carcinomas and pancreatic neuroendocrine tumours are clear exceptions to the rule, displaying an extensive transformation of their genomic landscape in advanced stages. Exposure to treatment further scars the tumour genome and introduces an evolutionary bottleneck that selects for known therapy-resistant drivers in approximately half of treated patients. Our data showcase the potential of pan-cancer whole-genome analysis to identify distinctive features of late-stage tumours and provide a valuable resource to further investigate the biological basis of cancer and resistance to therapies.

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.

The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.

Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

Lineage tracing of human development through somatic mutations.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

A survey of chromosomal instability measures across mechanistic models.

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

High karyotypic complexity is an independent prognostic factor in patients with CLL treated with venetoclax combinations.

Complex karyotypes have been associated with inferior outcomes in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy (CIT), whereas their prognostic impact in the context of venetoclax-based treatments is still debated. In this prospective analysis on karyotype complexity in CLL, we evaluated the impact of complex (≥3 chromosomal aberrations [CAs], CKTs) and highly complex karyotypes (≥5 CAs; hCKTs) as well as specific aberrations in previously untreated patients without TP53 aberrations undergoing either CIT or time-limited venetoclax-based therapies in the phase 3 GAIA/CLL13 trial. Karyotype analyses were available for 895 of 926 patients (96.7%), of whom 153 (17%) had a CKT and 43 (5%) hCKT. In the CIT arm, CKT was associated with shorter progression-free survival (PFS) (hazard ratio [HR] 2.58; 95% confidence interval [95% CI], 1.54-4.32; P < .001) and overall survival (HR, 3.25; 95% CI, 1.03-10.26; P = .044). In the pooled venetoclax arms, a multivariable analysis identified hCKTs (HR, 1.96; 95% CI, 1.03-3.72; P = .041), but not CKTs, as independent adverse prognosticators for PFS. The presence of translocations (unbalanced and/or balanced) was also independently associated with shorter PFSs in the venetoclax arms. CIT led to the acquisition of additional CAs (mean CAs, 2.0-3.4; from baseline to CLL progression), whereas karyotype complexity remained stable after venetoclax-based treatments (2.0, both time points). This analysis establishes highly complex karyotypes and translocations as adverse prognostic factors in the context of venetoclax-based combination treatments. The findings of this study support the incorporation of karyotyping into the standard diagnostic workup of CLL, because it identifies patients at high risk of poor treatment outcomes and thereby improves prognostication. This trial was registered at www.clinicaltrials.gov as #NCT02950051.

Primary cell cultures from the single-chromosome ant Myrmecia croslandi.

The number of chromosomes varies tremendously across species. It is not clear whether having more or fewer chromosomes could be advantageous. The probability of non-disjunction should theoretically decrease with smaller karyotypes, but too long chromosomes should enforce spatial constraint for their segregation during the mitotic anaphase. Here, we propose a new experimental cell system to acquire novel insights into the mechanisms underlying chromosome segregation. We collected the endemic Australian ant Myrmecia croslandi, the only known species with the simplest possible karyotype of a single chromosome in the haploid males (and one pair of chromosomes in the diploid females), since males are typically haploid in hymenopteran insects. Five colonies, each with a queen and a few hundreds of workers, were collected in the Canberra district (Australia), underwent karyotype analysis to confirm the presence of a single pair of chromosomes in worker pupae, and were subsequently maintained in the laboratory in Paris (France). Starting from dissociated male embryos, we successfully conducted primary cell cultures comprised of single-chromosome cells. This could be developed into a unique model that will be of great interest for future genomic and cell biology studies related to mitosis.

Prenatal diagnosis and genetic analysis of small supernumerary marker chromosomes in the eastern chinese han population: A retrospective study of 36 cases.

BACKGROUND: Small supernumerary marker chromosomes (sSMCs) are additional chromosomes with unclear structures and origins, and their correlations with clinical fetal phenotypes remain incompletely understood, which reduces the accuracy of genetic counseling. METHODS: We conducted a retrospective analysis of a cohort of 36 cases of sSMCs diagnosed in our center. We performed G-banding and chromosomal microarray analysis (CMA). The resulting karyotypes were compared with case reports in the literature and various databases including OMIM, DECIPHER, ClinVar, ClinGen, ISCA, DGV, and PubMed. RESULTS: Karyotype analysis data revealed that 19 out of 36 fetuses were mosaic. Copy number variants (CNVs) analysis results showed that 27 out of 36 fetuses harbored pathogenic/likely pathogenic variants. Among these 27 cases, 11 fetuses carried sex chromosome-related CNVs, including 4 female cases exhibiting Turner syndrome phenotypes and 7 cases showing Y chromosome deletions. In the remaining 16 fetuses with autosomal CNVs, 9 fetuses carried variants associated with Cat eye syndrome, Emanuel syndrome, Tetrasomy 18p, and 15q11-q13 duplication syndrome. Among these, 22 fetuses were terminated, and the remaining 5 fetuses were delivered and developed normally. Additionally, we identified a few variants with unclear pathogenicity. CONCLUSION: Cytogenetic analysis is essential for identifying the pathogenicity of sSMCs and increasing the accuracy of genetic counseling.

Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations.

The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen.

The causes and consequences of polyploidy in normal development and cancer.

Although nearly all mammalian species are diploid, whole-genome duplications occur in select mammalian tissues as part of normal development. Such programmed polyploidization involves changes in the regulatory pathways that normally maintain the diploid state of the mammalian genome. Unscheduled whole-genome duplications, which lead primarily to tetraploid cells, also take place in a substantial fraction of human tumors and have been proposed to constitute an important step in the development of cancer aneuploidy. The origins of these polyploidization events and their consequences for tumor progression are explored in this review.

Karyotypes of water frogs from the Pelophylax esculentus complex: results of cross-species chromosomal painting.

Amphibian species have the largest genome size enriched with repetitive sequences and relatively similar karyotypes. Moreover, many amphibian species frequently hybridize causing nuclear and mitochondrial genome introgressions. In addition, hybridization in some amphibian species may lead to clonality and polyploidization. All such events were found in water frogs from the genus Pelophylax. Among the species within the genus Pelophylax, P. esculentus complex is the most widely distributed and well-studied. This complex includes two parental species, P. ridibundus and P. lessonae, and their hybrids, P. esculentus, reproducing hemiclonally. Parental species and their hybrids have similar but slightly polymorphic karyotypes, so their precise identification is still required. Here, we have developed a complete set of 13 chromosome painting probes for two parental species allowing the precise identification of all chromosomes. Applying chromosomal painting, we identified homologous chromosomes in both parental species and orthologous chromosomes in their diploid hemiclonal hybrids. Comparative painting did not reveal interchromosomal exchanges between the studied water frog species and their hybrids. Using cross-specific chromosome painting, we detected unequal distribution of the signals along chromosomes suggesting the presence of species-specific tandem repeats. Application of chromosomal paints to the karyotypes of hybrids revealed differences in the intensity of staining for P. ridibundus and P. lessonae chromosomes. Thus, both parental genomes have a divergence in unique sequences. Obtained chromosome probes may serve as a powerful tool to unravel chromosomal evolution in phylogenetically related species, identify individual chromosomes in different cell types, and investigate the elimination of chromosomes in hybrid water frogs.

Increased genome size is caused by heterochromatin addition in two non-related bat species, Hesperoptenus doriae and Philetor brachypterus (Vespertilionidae, Chiroptera, Mammalia).

The average genome size (GS) of bats, which are the only mammals capable of powered flight, is approximately 18% smaller than that of closely related mammalian orders. The low nuclear DNA content of Chiroptera is comparable to that of birds, which are also characterized by a high metabolic rate. Only a few chiropteran taxa possess notable amounts of constitutive heterochromatin. Here, we studied the karyotypes of two non-related vesper bat species with unusually high amounts of constitutive heterochromatin: Hesperoptenus doriae and Philetor brachypterus. Conventional staining methods and whole-chromosome painting with probes derived from Myotis myotis (2n = 44), showing a karyotype close to that of the presumed ancestor of Vespertilionidae, revealed Robertsonian fusions as the main type of rearrangement leading to the exceptionally reduced diploid chromosome number of 2n = 26 in both species. Moreover, both karyotypes are characterized by large blocks of pericentromeric heterochromatin composed of CMA-positive and DA-DAPI-positive segments. In H. doriae, the heterochromatin accumulation has resulted in a genome size of 3.22 pg (1C), which is 40% greater than the mean genome size for the family. For P. brachypterus, a genome size of 2.94 pg was determined, representing an increase of about 28%. Most notably, in H. doriae, the presence of additional constitutive heterochromatin correlates with an extended mitotic cell cycle duration in vitro. A reduction in diploid chromosome number to 30 or lower is discussed as a possible cause of the accumulation of pericentromeric heterochromatin in Vespertilionidae.

Optical genome mapping enables constitutional chromosomal aberration detection.

Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."

Next-generation cytogenetics: Comprehensive assessment of 52 hematological malignancy genomes by optical genome mapping.

Somatic structural variants (SVs) are important drivers of cancer development and progression. In a diagnostic set-up, especially for hematological malignancies, the comprehensive analysis of all SVs in a given sample still requires a combination of cytogenetic techniques, including karyotyping, FISH, and CNV microarrays. We hypothesize that the combination of these classical approaches could be replaced by optical genome mapping (OGM). Samples from 52 individuals with a clinical diagnosis of a hematological malignancy, divided into simple (<5 aberrations, n = 36) and complex (≥5 aberrations, n = 16) cases, were processed for OGM, reaching on average: 283-fold genome coverage. OGM called a total of 918 high-confidence SVs per sample, of which, on average, 13 were rare and >100 kb. In addition, on average, 73 CNVs were called per sample, of which six were >5 Mb. For the 36 simple cases, all clinically reported aberrations were detected, including deletions, insertions, inversions, aneuploidies, and translocations. For the 16 complex cases, results were largely concordant between standard-of-care and OGM, but OGM often revealed higher complexity than previously recognized. Detailed technical comparison with standard-of-care tests showed high analytical validity of OGM, resulting in a sensitivity of 100% and a positive predictive value of >80%. Importantly, OGM resulted in a more complete assessment than any previous single test and most likely reported the most accurate underlying genomic architecture (e.g., for complex translocations, chromoanagenesis, and marker chromosomes). In conclusion, the excellent concordance of OGM with diagnostic standard assays demonstrates its potential to replace classical cytogenetic tests as well as to rapidly map novel leukemia drivers.

Targeted long-read sequencing identifies missing disease-causing variation.

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

Tri-ing to decipher trisomy AML.

Lam et al. compared trisomy acute myeloid leukaemia (AML) patients (inclusive of single trisomy, double trisomy or tetrasomy cases) with cytogenetically normal AML to uncover distinguishing molecular and prognostic features of trisomy AML. The study contributes to our understanding of trisomy AML, but the heterogeneity of trisomy subtypes remains a barrier to its study. Commentary on: Lam et al. Distinct karyotypic and mutational landscape in trisomy AML. Br J Haematol 2024;204:939-944.

Spontaneous chromosome instability and tissue culture-induced karyotypic alteration in wheat-Thinopyrum intermedium alien addition lines.

MAIN CONCLUSION: Wheat lines harboring wild-relative chromosomes can be karyotypically unstable during long-term maintenance. Tissue culture exacerbates chromosomal instability but appears inefficient to induce somatic homoeologous exchange between alien and wheat chromosomes. We assessed if long-term refrigerator storage with regular renewal via self-fertilization, a widely used practice for crop germplasm maintenance, would ensure genetic fidelity of alien addition lines, and explored the possibility of inducing somatic homoeologues exchange by tissue culture. We cytogenetically characterized sampled stock seeds of originally confirmed 12 distinct wheat-Thinopyrum intermedium alien addition lines (dubbed TAI lines), and subjected immature embryos of the TAI lines to tissue culture. We find eight of the 12 TAI lines were karyotypically departed from their original identity as bona fide disomic alien addition lines due to extensive loss of whole-chromosomes of both Th. intermedium and wheat origins during the ca. 3-decade storage. Rampant numerical chromosome variations (NCVs) involving both alien and wheat chromosomes were detected in regenerated plants of all 12 studied TAI lines, but at variable rates among the wheat sub-genomes and chromosomes. Compared with NCVs, structural chromosome variations (SCVs) occurred at substantially lower rates, and no SCV involving the added alien chromosomes was observed. The NCVs manifested only moderate effects on phenotypes of the regenerated plants under field conditions.

Prospective Investigation of Optical Genome Mapping for Prenatal Genetic Diagnosis.

BACKGROUND: Optical genome mapping (OGM) is a novel assay for detecting structural variants (SVs) and has been retrospectively evaluated for its performance. However, its prospective evaluation in prenatal diagnosis remains unreported. This study aimed to prospectively assess the technical concordance of OGM with standard of care (SOC) testing in prenatal diagnosis. METHODS: A prospective cohort of 204 pregnant women was enrolled in this study. Amniotic fluid samples from these women were subjected to OGM and SOC testing, which included chromosomal microarray analysis (CMA) and karyotyping (KT) in parallel. The diagnostic yield of OGM was evaluated, and the technical concordance between OGM and SOC testing was assessed. RESULTS: OGM successfully analyzed 204 cultured amniocyte samples, even with a cell count as low as 0.24 million. In total, 60 reportable SVs were identified through combined OGM and SOC testing, with 22 SVs detected by all 3 techniques. The diagnostic yield for OGM, CMA, and KT was 25% (51/204), 22.06% (45/204), and 18.14% (37/204), respectively. The highest diagnostic yield (29.41%, 60/204) was achieved when OGM and KT were used together. OGM demonstrated a concordance of 95.56% with CMA and 75.68% with KT in this cohort study. CONCLUSIONS: Our findings suggest that OGM can be effectively applied in prenatal diagnosis using cultured amniocytes and exhibits high concordance with SOC testing. The combined use of OGM and KT appears to yield the most promising diagnostic outcomes.