Targeted Capture Sequencing Research Articles

Direct genome sequencing of respiratory viruses from low viral load clinical specimens using the target capture sequencing technology.

The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging-especially in the case of respiratory viruses-due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2,000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/µL of SARS-CoV-2 or 625.1 copies/µL of influenza A virus. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/µL, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.IMPORTANCETarget capture sequencing has been developed and applied for direct genome sequencing of viruses in clinical specimens to overcome the low detection sensitivity of metagenomic next-generation sequencing. In this study, we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A, focusing on clinical specimens containing low copy numbers of viral genomes. Our results showed that the target capture sequencing yielded dramatically higher read counts than metagenomic sequencing for both viruses. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with other viruses, suggesting that this approach will not only detect a wide range of viruses but also contribute to epidemiological studies.

Evaluating the Efficacy of Target Capture Sequencing for Genotyping in Cattle.

(1) Background: Target capture sequencing (TCS) is potentially a cost-effective way to detect single-nucleotide polymorphisms (SNPs) and an alternative to SNP array-based genotyping. (2) Methods: We evaluated the effectiveness and reliability of TCS in cattle breeding scenarios using 48 female and 8 male samples. DNA was extracted from blood samples, targeted for 71,746 SNPs with TWIST probes, and sequenced on an MGI platform. GATK and BCFtools were evaluated for the best genotyping calling tool. The genotypes were compared to existing genotypes from the Versa50K SNP array of the same animals by measuring accuracy as concordance (%) and R2. (3) Results: In this study, 71,553 SNPs and 166 indels were identified. The genotype comparison of 37,130 common SNPs between TCS and SNP arrays yielded high agreement, with a mean concordance of 98%, R2 of 0.98 and Cohen's kappa of 0.97. The concordances of sex prediction, parent verification and validation of five genotype markers of interest important for Wagyu breeding were 100% between TCS and SNP array. The elements of the genomic relationship matrix (GRM) constructed from the SNP array and TCS data demonstrated a correlation coefficient approaching unity (r = 0.9998). (4) Conclusions: Compared to the SNP array, TCS is a comparable, cost-effective and flexible platform for genotyping SNPs, including non-model organisms and underrepresented commercial animal populations.

Development and validation of a 5K low-density SNP chip for Hainan cattle

BackgroundThis study aimed to design and develop a 5K low-density liquid chip for Hainan cattle utilizing targeted capture sequencing technology. The chip incorporates a substantial number of functional single nucleotide polymorphism (SNP) loci derived from public literature, including SNP loci significantly associated with immunity, heat stress, meat quality, reproduction, and other traits. Additionally, SNPs located in the coding regions of immune-related genes from the Bovine Genome Variation Database (BGVD) and Hainan cattle-specific SNP loci were included.ResultsA total of 5,293 SNPs were selected, resulting in 9,837 DNA probes with a coverage rate of 85.69%, thereby creating a Hainan cattle-specific 5K Genotyping by Target Sequencing (GBTS) liquid chip. Evaluation with 152 cattle samples demonstrated excellent clustering performance and a detection rate ranging from 96.60 to 99.07%, with 94.5% of SNP sites exhibiting polymorphism. The chip achieved 100% gender coverage and displayed a heterozygosity rate between 14.20% and 29.65%, with a repeatability rate of 99.65–99.85%. Analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed the potential regulatory roles of exonic SNPs in immune response pathways.ConclusionThe development and validation of the 5K GBTS liquid chip for Hainan cattle represent a valuable tool for genome analysis and genetic diversity assessment. Furthermore, it facilitates breed identification, gender determination, and kinship analysis, providing a foundation for the efficient utilization and development of local cattle genetic resources.

Emphasis on the importance of comprehensive clinical and genetic analysis - spinal muscular atrophy combined with phenylketonuria: A case report.

Both spinal muscular atrophy (SMA) and Phenylketonuria (PKU) are caused by biallelic pathogenic mutations. However, there has been no report on case who suffering from both diseases simultaneously. SMA mainly affects the motor function while PKU may have an impact on both the intelligence and motor function. But if only 1 disease is treated while neglecting the other, the treatment effect will be compromised. Here, for the first time, we report a case from China diagnosed with both these diseases and treated properly. A boy was admitted to the Children's Hospital Affiliated to Shandong University (Jinan, China) due to "limb weakness for 19 months" when he was 22 months old. Considering that the child's motor function development is delayed, we made a comprehensive examinations including inherited metabolic diseases and found a significantly increase of phenylalanine concentration in the blood which indicating PKU. Combined with his typical clinical manifestations of SMA, target capture sequencing followed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) technologies were used for genetic confirmation. SMA and PKU was confirmed. The child was treated with risdiplam and low phenylalanine formula immediately when he was diagnosed with both SMA and PKU. The child showed remarkable improvement in motor function and significant decrease of blood phenylalanine concentration after treatment. To our knowledge, this is the first reported case of SMA combined with PKU. This case expands our understanding of diagnosis for synchronous SMA and PKU and highlights the importance of comprehensive examinations and the utilizing of various genetic testing methods to make an accurate diagnosis of genetic diseases, which may help avoiding the progressive damage caused by certain genetic disease with insidious clinical symptoms.

Genomic incongruence accompanies the evolution of flower symmetry in Eudicots: a case study in the poppy family (Papaveraceae, Ranunculales).

Reconstructing evolutionary trajectories and transitions that have shaped floral diversity relies heavily on the phylogenetic framework on which traits are modelled. In this study, we focus on the angiosperm order Ranunculales, sister to all other eudicots, to unravel higher-level relationships, especially those tied to evolutionary transitions in flower symmetry within the family Papaveraceae. This family presents an astonishing array of floral diversity, with actinomorphic, disymmetric (two perpendicular symmetry axes), and zygomorphic flowers. We generated nuclear and plastid datasets using the Angiosperms353 universal probe set for target capture sequencing (of 353 single-copy nuclear ortholog genes), together with publicly available transcriptome and plastome data mined from open-access online repositories. We relied on the fossil record of the order Ranunculales to date our phylogenies and to establish a timeline of events. Our phylogenomic workflow shows that nuclear-plastid incongruence accompanies topological uncertainties in Ranunculales. A cocktail of incomplete lineage sorting, post-hybridization introgression, and extinction following rapid speciation most likely explain the observed knots in the topology. These knots coincide with major floral symmetry transitions and thus obscure the order of evolutionary events.

A consistency comparison between next-generation sequencing and the FISH method for gene rearrangement detection in B-cell lymphomas

Objective: To compare the consistency of lymphoma multigene detection panels based on next-generation sequencing (NGS) with FISH detection of B-cell lymphoma gene rearrangement. Methods: From January 2019 to May 2023, fusion genes detected by lymphoma-related 413 genes that targeted capture sequencing of 489 B-cell lymphoma tissues embedded in paraffin were collected from Henan Cancer Hospital, and the results were compared with simultaneous FISH detection of four break/fusion genes: BCL2, BCL6, MYC, and CCND1. Consistency was defined as both methods yielding positive or negative results for the same sample. The relationship between fusion mutation abundance in NGS and the positivity rate of cells in FISH was also analyzed. Results: Kappa consistency analysis revealed high consistency between NGS and FISH in detecting the four B-cell lymphoma-related gene rearrangement (P<0.001 for all) ; however, the detection rates of positive individuals differed for the four genes. Compared with FISH, NGS demonstrated a higher detection rate for BCL2 rearrangement, a lower detection rate for BCL6 and MYC rearrangement, and a similar detection rate for CCND1 rearrangement. No correlation was found between fusion mutation abundance in NGS and the positivity rate of cells in FISH. Conclusions: NGS and FISH detection of B-cell lymphoma gene rearrangement demonstrate overall good consistency. NGS is superior to FISH in detecting BCL2 rearrangement, inferior in detecting MYC rearrangement, and comparable in detecting CCND1 rearrangement.

Analysis of Breast Cancer Brain Metastases Reveals an Enrichment of Cyclin-Dependent Kinase 12 Structural Rearrangements in Human Epidermal Growth Factor Receptor 2-Positive Disease.

Genomic alterations have been identified in patients with breast cancer brain metastases (BCBMs), but large structural rearrangements have not been extensively studied. We analyzed the genomic profiles of 822 BCBMs and compared them with 11,988 local, breast-biopsied breast cancers (BCs) and 15,516 non-CNS metastases (Non-CNS M) derived from formalin-fixed paraffin-embedded material using targeted capture sequencing. Nine genes with structural rearrangements were more prevalent within BCBMs as compared with local BCs and Non-CNS M (adjusted-P < .05) and displayed a prevalence of >0.5%. The most common rearrangements within BCBMs involves cyclin-dependent kinase 12 (CDK12; 3.53%) as compared with the local BC (0.86%; adjusted-P = 7.1 × 10-8) and Non-CNS M specimens (0.68%; adjusted-P = 3.7 × 10-10). CDK12 rearrangements had a significantly higher frequency within human epidermal growth factor receptor 2 (HER2)-positive BCBMs (14.59%) compared with HER2-positive BCs (7.80%; P = 4.6 × 10-3) and HER2-positive Non-CNS M (7.87%; P = 4.8 × 10-3). The most common structural rearrangements involve CDK12 with the higher prevalence in HER2-positive BCBMs. These data support more detailed investigation of the role and importance of CDK12 rearrangements in BCBMs.

Assessing myBaits Target Capture Sequencing Methodology Using Short-Read Sequencing for Variant Detection in Oat Genomics and Breeding.

The integration of target capture systems with next-generation sequencing has emerged as an efficient tool for exploring specific genetic regions with a high resolution and facilitating the rapid discovery of novel alleles. Despite these advancements, the application of targeted sequencing methodologies, such as the myBaits technology, in polyploid oat species remains relatively unexplored. In this study, we utilized the myBaits target capture method offered by Daicel Arbor Biosciences to detect variants and assess their reliability for variant detection in oat genomics and breeding. Ten oat genotypes were carefully chosen for targeted sequencing, focusing on specific regions on chromosome 2A to detect variants. The selected region harbors 98 genes. Precisely designed baits targeting the genes within these regions were employed for the target capture sequencing. We employed various mappers and variant callers to identify variants. After the identification of variants, we focused on the variants identified via all variants callers to assess the applicability of the myBaits sequencing methodology in oat breeding. In our efforts to validate the identified variants, we focused on two SNPs, one deletion and one insertion identified via all variant callers in the genotypes KF-318 and NOS 819111-70 but absent in the remaining eight genotypes. The Sanger sequencing of targeted SNPs failed to reproduce target capture data obtained through the myBaits technology. Similarly, the validation of deletion and insertion variants via high-resolution melting (HRM) curve analysis also failed to reproduce target capture data, again suggesting limitations in the reliability of the myBaits target capture sequencing using short-read sequencing for variant detection in the oat genome. This study shed light on the importance of exercising caution when employing the myBaits target capture strategy for variant detection in oats. This study provides valuable insights for breeders seeking to advance oat breeding efforts and marker development using myBaits target capture sequencing, emphasizing the significance of methodological sequencing considerations in oat genomics research.

Plastid phylogenomics and cytonuclear discordance in Rubioideae, Rubiaceae.

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Development of 51 K liquid-phased probe array for Loblolly and Slash pines and its application to GWAS of Slash pine breeding population

Loblolly pine (Pinus taeda) and Slash pine (Pinus elliottii) are important economically and ecologically tree species worldwide. To implement genomic based breeding strategy, genotyping large number of samples is needed. However, huge and complex pines genome make it difficult to obtain abundant and cost-effectively markers. In this study, we designed a SNP array containing 51 K liquid-phased probes based on the published SNPs and probes of the two species, novel Slash pine-specific long-read transcript, and SNPs obtained from a double digest Restriction-Site Associated DNA sequencing (ddRADseq). We performed targeted capture sequencing using the developed SNP array on three pine species of Loblolly, Slash, and Caribbean pine (Pinus caribaea). The probe capture had an efficiency of 68.26% on average. Principal Component Analysis (PCA), phylogenetic analysis, and genetic structure analysis of 28,670 SNPs genotyped for the three pine species demonstrated the transferability of the SNP array for the three pines. Furthermore, we genotyped 221 range-wide Slash pine genotypes, analyzed their genetic diversity and performed a genome-wide association study (GWAS). We identified 95 SNPs significantly associated with growth and wood quality traits of Slash pine. We have identified potential roles of the MYB2 and MYB46 transcription factors in regulating cell cycle and differentiation in Slash pine, while also suggesting that AP2/ERF and Glycosyl hydrolase may play significant roles in wood formation processes. This new SNP array can be widely applied in future molecular breeding research of conifers.

Phylogenomics-driven host test list selection for weed biological control

A concern in weed biological control research is the potential for candidate biocontrol agents to impact not only the target weed but also native or economically important flora. The degree of evolutionary relatedness between the target weed species and a non-target species is a key predictor of the susceptibility of the non-target to the biocontrol agent. To manage this risk, biocontrol practitioners need to understand the phylogenetic position of the host weed relative to non-target plant species. However, comprehensively sampled phylogenetic trees are often unavailable, with incomplete information scattered across multiple publications. Further, older published phylogenies based on Sanger sequence data often lack branch resolution and support, which increases uncertainty in biocontrol decision making. Decreasing sequencing cost and technological advances have led to phylogenomic approaches being more widely used to understand evolutionary relationships between species. For example, target capture sequencing methods using bait kits such as Angiosperms353 enable cost-effective and timely phylogenomic-level analysis of flowering plant groups at different scales. Here, we introduce a workflow to embed a comprehensive understanding of evolutionary relationships into the efficient development of host test lists in weed biological control. We demonstrate the effectiveness of the workflow through a case study on the major crop weed flaxleaf fleabane (Erigeron bonariensis). Phylogenomic analysis was conducted on 280 species of the tribe Astereae (family Asteraceae) occurring in Australia and New Zealand, clarifying relationships between the target species and related clades of native and non-native Astereae. We consider the phylogenetic tree in the context of a previously proposed host test list and discuss taxonomic implications, highlighting avenues of future molecular-based work to uncover the origin of Australian fleabanes. This study provides a workflow and demonstrates the practical application of target sequence capture for phylogenomic inference to support risk analysis and decision making in classical weed biological control.

CtDNA improves prognostic prediction for patients with relapsed/refractory MM receiving ixazomib, lenalidomide, and dexamethasone

AbstractIt remains elusive how driver mutations, including those detected in circulating tumor DNA (ctDNA), affect prognosis in relapsed/refractory multiple myeloma (RRMM). Here, we performed targeted-capture sequencing using bone marrow plasma cells (BMPCs) and ctDNA of 261 RRMM cases uniformly treated with ixazomib, lenalidomide, and dexamethasone in a multicenter, prospective, observational study. We detected 24 and 47 recurrently mutated genes in BMPC and ctDNA, respectively. In addition to clonal hematopoiesis–associated mutations, varying proportion of driver mutations, particularly TP53 mutations (59.2% of mutated cases), were present in only ctDNA, suggesting their subclonal origin. In univariable analyses, ctDNA mutations of KRAS, TP53, DIS3, BRAF, NRAS, and ATM were associated with worse progression-free survival (PFS). BMPC mutations of TP53 and KRAS were associated with inferior PFS, whereas KRAS mutations were prognostically relevant only when detected in both BMPC and ctDNA. A total number of ctDNA mutations in the 6 relevant genes was a strong prognostic predictor (2-year PFS rates: 57.3%, 22.7%, and 0% for 0, 1, and ≥2 mutations, respectively) and independent of clinical factors and plasma DNA concentration. Using the number of ctDNA mutations, plasma DNA concentration, and clinical factors, we developed a prognostic index, classifying patients into 3 categories with 2-year PFS rates of 57.9%, 28.6%, and 0%. Serial analysis of ctDNA mutations in 94 cases revealed that TP53 and KRAS mutations frequently emerge after therapy. Thus, we clarify the genetic characteristics and clonal architecture of ctDNA mutations and demonstrate their superiority over BMPC mutations for prognostic prediction in RRMM. This study is a part of the C16042 study, which is registered at www.clinicaltrials.gov as #NCT03433001.

Target enrichment sequencing coupled with GWAS identifies MdPRX10 as a candidate gene in the control of budbreak in apple.

The timing of floral budbreak in apple has a significant effect on fruit production and quality. Budbreak occurs as a result of a complex molecular mechanism that relies on accurate integration of external environmental cues, principally temperature. In the pursuit of understanding this mechanism, especially with respect to aiding adaptation to climate change, a QTL at the top of linkage group (LG) 9 has been identified by many studies on budbreak, but the genes underlying it remain elusive. Here, together with a dessert apple core collection of 239 cultivars, we used a targeted capture sequencing approach to increase SNP resolution in apple orthologues of known or suspected A. thaliana flowering time-related genes, as well as approximately 200 genes within the LG9 QTL interval. This increased the 275 223 SNP Axiom® Apple 480K array dataset by an additional 40 857 markers. Robust GWAS analyses identified MdPRX10, a peroxidase superfamily gene, as a strong candidate that demonstrated a dormancy-related expression pattern and down-regulation in response to chilling. In-silico analyses also predicted the residue change resulting from the SNP allele associated with late budbreak could alter protein conformation and likely function. Late budbreak cultivars homozygous for this SNP allele also showed significantly up-regulated expression of C-REPEAT BINDING FACTOR (CBF) genes, which are involved in cold tolerance and perception, compared to reference cultivars, such as Gala. Taken together, these results indicate a role for MdPRX10 in budbreak, potentially via redox-mediated signaling and CBF gene regulation. Moving forward, this provides a focus for developing our understanding of the effects of temperature on flowering time and how redox processes may influence integration of external cues in dormancy pathways.

Abstract B001: Does deep intronic splicing in breast cancer susceptibility genes contribute to breast cancer?

Abstract Breast cancer (BC) is the most common cancer and leading cause of cancer mortality in women. Average lifetime BC risk for a woman in the United States is 13%. However, pathogenic variants in BC susceptibility genes such as BRCA1/2, ATM, and RAD51C, or high polygenic risk scores substantially increase BC risk. Women with a family history of BC and/or ovarian cancer, BC &amp;lt; age 40, or BC and second primary tumor are considered high-risk with an increased likelihood of having a pathogenic variant in a BC susceptibility gene. Yet, more than 60% of women with high-risk BC test negative for coding variants. In these women, possible causal variation could be non-coding in deep intronic or regulatory regions, or cryptic structural variants (SV). To determine whether deep intronic splicing contributes to BC in high-risk women testing negative for pathogenic coding variants in BC genes, we analyzed the intronic sequences of 649 BRCA1/BRCA2 negative individuals with high-risk BC. The samples had undergone targeted capture sequencing of exons and introns of more than 100 target genes including ATM and RAD51C. From the 649 samples, we identified three intronic splicing variants in ATM and RAD51C in 10 samples (1.5%) that had SpliceAI delta scores &amp;gt;0.5 suggesting changes to splicing. One splicing donor gain variant, RAD51C c.145+246A&amp;gt;T, was found exclusively in women of self-reported African ancestry at a frequency of 0.047. In gnomAD, the variant was found exclusively in individuals of African ancestry at a frequency of 0.012. A 1.3-fold enrichment in variant frequency was observed in AFR women under 65 with breast cancer compared to age-matched cancer free controls in the All of Us dataset. Using lymphoblastoid-cell derived RNA from carriers of RAD51C c.145+246A&amp;gt;T, we performed RT-PCR and cDNA amplification targeting the exons surrounding the variant and observed a lengthened exon. Targeted amplicon sequencing also confirmed the presence of intronic sequence in the variant cDNA. The introduced intronic sequence contains a UGA stop codon 12 bp from the end of the exon which could led to loss of RAD51C function. We have created minigene vectors in the RHCglo background and are performing minigene splicing assays to validate the splicing changes in ATM due to intronic variants. Gene functional assays and association studies will be conducted to determine the functional effects and risk conferred by these variants. Understanding the level of contribution of deep intronic splicing in known BC susceptibility genes is critically important, as results impact sequencing offered to patients, and if positive, their medical management. Citation Format: Mwangala P. Akamandisa, Bradley Wubbenhorst, Kurt D'Andrea, Heather Symecko, Rhonda Gillette, Payal D. Shah, Angela R. Bradbury, Kara N. Maxwell, Susan M. Domchek, Katherine L. Nathanson. Does deep intronic splicing in breast cancer susceptibility genes contribute to breast cancer? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B001.

GenoBaits®WheatplusEE: a targeted capture sequencing panel for quick and accurate identification of wheat-Thinopyrum derivatives.

A total of 90,000 capture probes derived from wheat and Thinopyrum elongatum were integrated into one chip, which served as an economical genotype for explorating Thinopyrumspecies and their derivatives. Thinopyrum species play a crucial role as a source of new genetic variations for enhancing wheat traits, including resistance to both abiotic and biotic factors. Accurate identification of exogenous chromosome(s) or chromosome segments or genes is essential following the introduction of alien genetic material into wheat, but this task remains challenging. This study aimed to develop a high-resolution wheat-Thinopyrum elongatum array, named GenoBaits®WheatplusEE, to trace alien genetic information by genotyping using a target sequencing system. This GenoBaits®WheatplusEE array included 90,000 capture probes derived from two species and integrated into one chip, with 10,000 and 80,000 originating from wheat and Th. elongatum, respectively. The capture probes were strategically positioned in genes and evenly distributed across the genome, facilitating the development of a roadmap for identifying each alien gene. The array was applied to the high-throughput identification of the alien chromosomes or segments in Thinopyrum and distantly related species and their derivatives. Our results demonstrated that the GenoBaits®WheatplusEE array could be used for direct identification of the breakpoint of alien segments, determine copy number of alien chromosomes, and reveal variations in wheat chromosomes by a single round of target sequencing of the sample. Additionally, we could efficiently and cost-effectively genotype, supporting the exploration of subgenome composition, phylogenetic relationships, and polymorphisms in essential genes (e.g., Fhb7 gene) among Thinopyrum species and their derivatives. We hope that GenoBaits®WheatplusEE will become a widely adopted tool for exporting wild germplasm for wheat improvement in the future.

Identification of Superior Haplotypes and Haplotype Combinations for Grain Size- and Weight-Related Genes for Breeding Applications in Rice (Oryza sativa L.).

The identification of superior haplotypes and haplotype combinations is essential for haplotype-based breeding (HBB), which provides selection targets for genomics-assisted breeding. In this study, genotypes of 42 functional genes in rice were analyzed by targeted capture sequencing in a panel of 180 Indica rice accessions. In total, 69 SNPs/Indels in seven genes were detected to be associated with grain length (GL), grain width (GW), ratio of grain length-width (L/W) and thousand-grain weight (TGW) using candidate gene-based association analysis, including BG1 and GS3 for GL, GW5 for GW, BG1 and GW5 for L/W, and AET1, SNAC1, qTGW3, DHD1 and GW5 for TGW. Furthermore, two haplotypes were identified for each of the seven genes according to these associated SNPs/Indels, and the amount of genetic variation explained by different haplotypes ranged from 3.24% to 27.66%. Additionally, three, three and eight haplotype combinations for GL, L/W and TGW explained 25.38%, 5.5% and 22.49% of the total genetic variation for each trait, respectively. Further analysis showed that Minghui63 had the superior haplotype combination Haplotype Combination 4 (HC4) for TGW. The most interesting finding was that some widely used restorer lines derived from Minghui63 also have the superior haplotype combination HC4, and our breeding varieties and lines using the haplotype-specific marker panel also confirmed that the TGW of the lines was much higher than that of their sister lines without HC4, suggesting that TGW-HC4 is the superior haplotype combination for TGW and can be utilized in rice breeding.

Integrated Analysis of KIT Exon 17 Mutations and Flow-MRD Refines Risk Stratification in Pediatric Acute Myeloid Leukemia with RUNX1:: RUNX1T1

INTRODUCTION: Pediatric acute myeloid leukemia (AML) with RUNX1:: RUNX1T1 is considered a favorable risk group, with a high probability of event-free survival (EFS) in recent clinical trials. However, 20-30% of patients still experience relapse within three years and some patients have poor outcome. Several prognostic factors, including secondary genetic abnormalities and treatment response, have been previously reported. Flow cytometry-based measurable residual disease (flow-MRD) has been suggested as a poor prognostic factor in AML with RUNX1:: RUNX1T1. Additionally, KIT mutations, particularly mutations of exon 17, have been associated with poor EFS in both adult and pediatric patients. However, the combined impact of KIT exon 17 mutations and flow-MRD on prognosis has not been thoroughly investigated. This study aims to evaluate the clinical and genetic features of pediatric patients with RUNX1:: RUNX1T1-positive AML enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-12 trial and determine if integrated analysis of flow-MRD and KIT exon 17 mutations can improve risk stratification. METHOD: A total of 101 pediatric AML patients with RUNX1:: RUNX1T1 were enrolled in the study. The patients were treated in the JPLSG AML-12 trial, and their clinical and genetic characteristics were analyzed. Targeted capture sequencing was performed on bone marrow samples obtained at diagnosis. The custom gene panel was designed for mutation profiling of pediatric AML, including 504 genes. Flow-MRD was centrally monitored at the end of induction 1 (EOI1) and the end of induction 2 but was not used to guide subsequent therapies. Leukemia-associated immunophenotypes were identified in diagnostic specimens and marker combinations that allowed detection of leukemia with a sensitivity of at least 0.1% were applied to subsequent samples. RESULTS: Patients with KIT exon 17 mutations had a higher white blood cell count at diagnosis and a significantly higher rate of MRD positivity (≥0.1%) at EOI1 compared to patients without KIT exon 17 mutations. Immunophenotypic analysis showed no significant differences in CD117 or CD33 expression between patients with and without KIT exon 17 mutations; whereas CD19 expression was observed less frequently and, although not significantly, CD56 expression was observed more frequently in the patients with KIT exon 17 mutations. The genetic landscape analysis revealed that KIT mutations were the most common and tended to coexist with ARID1B mutations, while NRAS mutations were mutually exclusive. The 5-year EFS and overall survival (OS) for the entire cohort were 67.3% and 82.7%, respectively. Negative MRD at EOI1 was associated with significantly superior 5-year EFS and OS (71.6% and 85.9%) compared to positive MRD (12.5% and 37.5%)(p&amp;lt;0.01). Patients with KIT exon 17 mutations had significantly inferior 5-year EFS and OS (46.9% and 62.2%) compared to those without KIT exon 17 mutations (76.8% and 92.4%) (p&amp;lt;0.01). Furthermore, we evaluated a prognostic impact of MRD within the KIT exon 17 mutated and non-mutated cases. As for the non-mutated cases, the 5-year OS in the patients with negative MRD was 92.2%. Among the KIT-mutated cases, the 5-year OS in the patients with negative and positive MRD was 68.0% and 33.3%, respectively. CONCLUSION: Patients with both non-mutated KIT exon 17 and negative MRD have the best prognosis, while positive MRD and KIT exon 17 mutations are associated with poorer outcomes. These findings indicated that integrated analysis of flow-MRD and KIT exon 17 status enables optimal risk assignment strategies in pediatric AML with RUNX1:: RUNX1T1.

Prognostic Impact of Chromosomal Abnormalities in TP53-Mutated Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndromes

Background: Background: Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) exhibit clinical heterogeneity, and genomic profiles play a crucial role in disease characteristics and survival outcomes. Elli Papaemmanuil et al. classified AML into 11 genomic subgroups (NEJM, 2016), where TP53 mutations and aneuploidy predicted dismal survival. Cases with TP53 mutations are often accompanied by complex chromosomal abnormalities (CAs), but the prognostic impact and target genes of CAs have not been fully studied. Methods: To clarify this issue, specimens of AML and MDS with excess blasts (MDS-EB) were collected from 1,001 patients enrolled at collaborating institutes of Kyoto Hematology Study Group between July 2017 and January 2023. Targeted capture sequencing with 1,216 single nucleotide polymorphism sites was used to analyze primary samples, allowing assessment of copy number alteration (CNA) profiles. The target gene panel included common mutations in myeloid neoplasms. Mutation calling was performed using the established pipeline Genomon 2. Prognostic impact of CNA, single nucleotide variants, and insertion/deletion with 10% or more frequency was evaluated, and the frequencies of CNA were calculated for the short and long arms of chromosomes 1-22 and the X chromosome. Univariate and multivariate analyses were conducted using statistical significance criteria of p &amp;lt; 0.05. Results: The average sequencing depth was 548. Among 1,001 patients, 224 (22.4%) harbored TP53 mutations. TP53-mutated cases had a median age of 70 (24-95) years old at diagnosis, with 117 (52.2%) being AML and 107 (47.8%) MDS-EB. TP53 biallelic mutation, defined by 2 or more mutations in TP53 or loss of heterogeneity of TP53 locus, was present in 200 (89.3%) of cases, more commonly associated with 3 or more CAs or CNA (complex karyotype (CK)-like) than monoallelic mutation (99% vs. 42%, p = 1.9x10 -13). The most frequent affected lesions were del(5q), del(7q), del(7p), del(12p), del(18q), gain(11q), and gain(21q) with frequencies of 76%, 59%, 34%, 33%, 28%, 27%, and 26%, respectively. In survival analysis, gain(21q), gain(19p), del(16q), del(3q), del(19p), del(13q), and del(4q), along with diagnosis of AML ( p = 2.0x10 -2), TP53 biallelic mutation ( p = 6.8x10 -3), and CK-like ( p = 3.4x10 -3), were associated with poor outcomes in univariate analysis. Multivariate analysis revealed gain(19p), del(3q), del(13q), del(4q), diagnosis of AML, and CK-like as independent poor prognostic factors. Here we have defined gain(19p), del(3q), del(13q), and del(4q) as “Risk CNA”. A significant difference in outcomes was observed between patients with and without Risk CNA (n = 110 and n = 114, respectively, Hazard ratio = 2.6, p = 6.0x10 -8): The 6-month survival rate was 70 (95% Confidence interval [60-79]) % for patients without Risk CNA and 42 [31-52] % for those with Risk CNA. Patients with two or more Risk CNAs had worse prognosis than those with one ( p = 2.7x10 -2), indicating independent or additive contributions to outcomes. Similar trends were observed when limiting the analysis to TP53-biallelic cases. Further investigation of Risk CNA target genes was performed by identifying commonly amplified/deleted lesions. Target genes of gain(19p) included DNM2, CARM1, SMARCA4, LDLR, and EPOR, while RB1 and TET2 were potential candidates of del(13q) and del(4q), respectively. Identification of candidate genes for del(3q) was challenging due to broad common deleted lesions. Conclusion: Chromosomal abnormalities stratify the prognosis of AML and MDS-EB with TP53 mutation. The number of Risk CNA also impacts survival. Investigation of target genes of Risk CNA suggests potential therapeutic targets for TP53-mutated myeloid neoplasms, warranting further study for treatment optimization.

Clonal Evolution of Der(1;7)(q10;p10) Myeloid Neoplasms

Background der(1;7)(q10;p10) (der(1;7)) is a recurrent unbalanced translocation found in 1.5~6% of MDS and AML patients. It is caused by rearrangement of centromere alpha satellites in chromosomes 1 and 7 resulting in an amplification of 1q and a deletion of 7q. Previous studies comparing der(1;7)(+) cases to those with other lesions of 7q loss (-7/del(7q)) demonstrated a higher frequency of RUNX1 mutations, no TP53 mutations and a better prognosis for der(1;7) cases suggesting that der(1;7)(+) may define a unique entity of myeloid neoplasms (MN). However, with the lack of comprehensive molecular characterization using unbiased platforms such as whole genome/exome sequencing, the entire picture of der(1;7)(+) MN still remains unclear. Methods We enrolled a total of 148 MN cases harboring der(1;7) and an additional 3,238 der(1;7)(−) MN cases: -7/del(7q), +1q and ‘OTHER’. We first analyzed paired tumor/normal DNA from 26 der(1;7) cases using whole exome sequencing (WES), followed by targeted-capture sequencing of 329 genes, including those commonly mutated in MN and others newly identified in der(1;7) cases in WES. To investigate the origin of der(1;7)(+) MN, we also enrolled 11,234 healthy individuals to evaluate der(1;7) in clonal hematopoiesis, in which der(1;7) was detected on based on SNP array-based karyotyping. Results In accordance with previous reports, der(1;7) MDS cases showed an improved overall survival (OS) compared to -7/del(7q) MDS cases (Hazard Ratio (HR) =0.71, p=0.098), which however, was shorter than OS in +1q (HR=1.36, p=0.11) and ‘OTHER’ (HR=1.8, p&amp;lt;0.001) MDS cases. der(1;7)(+) cases were characterized by very high mortality from infection (45.5%), while mortality from disease progression was less common (36.4%), showing a sharp contrast to -7/del(7q) and ‘OTHER’ MDS cases which had 13.9% and 10.8% infection-related deaths, and 72.3% and 76.9% disease progression, respectively (Figure 1A.). WES in 26 der(1;7)(+) cases revealed a novel transcription factor, MYB in 2 cases, which was found in 18 (12%) of 148 der(1;7) cases using targeted-capture sequencing. der(1;7)(+) cases were characterized by high frequency of mutations in transcription factors, including RUNX1 (37.8%), GATA2 (12%), ETV6 (13%), and BCOR (14%) (Figure 1B.), which were significantly more frequent in der(1;7) cases than other sub-types (Odds Ratio (OR)&amp;lt;6)(Figure 1B.). Very high frequency of ETNK1 mutations is highly unique to der(1;7)(+) cases (18.9%) compared to that in other cases (&amp;lt;10%). High frequency of mutations in transcription factors seems to be a unique feature of der(1;7)(+) cases. Trisomy 8 and del(20q) CNAs were also common in der(1;7) cases (16% and 21%, respectively). Together with DNMT3A and del(20q), der(1;7) showed the highest variant allele frequency (VAF), suggesting the early origin of these lesions. Additionally, we identified der(1;7) in 6 out of 11,234 healthy individuals using SNP array-based highly sensitive copy number detection, of which 4 died from MDS, suggesting that der(1;7)(+) clonal hematopoiesis might be the origin of MDS in these cases. Moreover, we also analyzed longitudinal samples from 9 der(1;7)(+) cases using duplex-sequencing, der(1;7) showed consistently high VAF, further supporting that der(1;7) tended to represent the major clone. Conclusion Showing a unique set of co-mutation pattern and clinical features, der(1;7) MN defines a distinct subset of myeloid neoplasms, which may originate from der(1;7)(+) clonal hematopoiesis. der(1;7)(+) patients were at higher risk of life-threatening infection, which needs to be carefully prevented and monitored during their clinical course.

TP53 and CDKN2A alterations Define a Poor Prognostic Subgroup in Patients with Nodal T-Follicular Helper Cell Lymphoma

Nodal T-follicular helper cell lymphoma (nTFHL) is a subset of peripheral T-cell lymphoma (PTCL) characterized by a distinct immunophenotype (such as CD10, BCL6, CXCL13, PD1, and ICOS) and mutations (including TET2, RHOA G17V, and IDH2 R172). However, the impact of molecular classification based on genetic profiling on prognosis in nTFHL remains largely unknown. Here we performed targeted-capture sequencing (targeted-seq) covering 235 T/NK-cell lymphoma-related genes with a mean depth of 526×. Then, we analyzed 49 genes which were reported as drivers in the previous PTCL studies to reveal the associations between clinical phenotypes and somatic alterations in a cohort of 153 patients whose clinical information was available and who were pathologically diagnosed with nTFHL according to the WHO-HAEM5. When mutation and copy number alteration (CNA) data were combined, we identified recurrent alterations in 40 among 49 driver genes. TET2 was most frequent (68%), followed by RHOA (51%), IDH2 (27%), DNMT3A (20%), PLCG1 (9%), TP53 (7%), KMT2C (6%), VAV1 (6%), CD28 (6%), KMT2D (6%), NOTCH1 (5%), and CDKN2A (5%). To create a molecular classification based on genetic alterations in nTFHL, we conducted hierarchical clustering using driver alterations and arm-level CNAs. This analysis identified four molecular subgroups named after their characteristic genetic alterations ( Fig. 1A). TR-ID (+) ( n = 52) and TR-ID (-) ( n = 60) were defined by TFH-related mutations ( TET2 and RHOA). Among them, TR-ID (+) harbored IDH2 and/or DNMT3A mutations. A53 ( n = 8) was notable for biallelic inactivation of TP53 and/or CDKN2A and associated with marked aneuploidy. NSD ( n = 33) had no subgroup-defining alterations with the fewest number of mutations. Clinically, TR-ID (+) tumors were mostly nTFHL-angioimmunoblastic-type (AI), which is consistent with the enrichment of IDH2 and/or DNMT3A mutations. On the other hand, TR-ID (-) tumors exhibited a higher proportion of nTFHL-NOS. NSD exhibited lower LDH levels and was significantly associated with younger age and localized stage than other subgroups. A53 showed the worst prognosis with a median overall survival (OS) of 15.1 months (mos) ( Fig. 1B). By contrast, the OS of NSD was significantly superior to the others (median OS not reached). The OS of TR-ID (+) and TR-ID (-) was comparable with a median OS of 40.6 and 46.4 mos, respectively. Notably, A53 remained significant in multivariate analyses adjusted for IPI or PIT score. Thus, genetic profiling enables to classify nTFHL patients into genetically and clinically distinct subgroups, including the unfavorable prognostic subgroup (A53). Next, to identify potential genes linked to the poor prognosis of the A53 subtype, we examined the prognostic impact of individual alterations. Among the genes examined, TP53 and CDKN2A, hallmarks of A53, were significantly associated with shorter OS in univariate analysis. Consequently, cases with TP53 and/or CDKN2A alterations showed a poor prognosis (median OS 11.4 vs 181.2 mos, P = 8.2×10 -7), thereby reinforcing the negative prognostic impact of A53. Then, we investigated other prognostic factors within each molecular subgroup. First, in TR-ID (+) and (-) cases, we examined the EBV status, which was known to be associated with worse prognosis in nTFHL-AI. Among them, 22 (16%) cases were EBV-positive. Despite no observed differences in genetic and clinical factors, EBV-positive cases exhibited a worse prognosis (median OS 66.5 vs 14.9 mos, P = 0.0018). Next, we assessed the influence of TCR clonality on prognosis in NSD cases. The clonal T-cell fraction assessed by targeted-seq was highest in A53 (median, 75%) but lowest in NSD (median, 16%). OS of NSD cases was further stratified by T-cell clonality (median OS not reached vs 67.7 mos for low and high clonal T-cell fraction; P = 0.042), while the presence of driver alterations failed to predict the prognosis. These findings suggest that assessment of T-cell clonality helps to identify the favorable prognostic subgroup characterized by low tumor content. In summary, we defined four molecular subgroups with different clinical features and also clarified subgroup-specific prognostic factors in nTFHL. Particularly, we identified the molecular subgroup with unfavorable prognosis (A53) for which different clinical approach should be warranted. This classification will refine the patient prognostication and stratification in nTFHL.