Allelic Bias Research Articles

Bayesian Estimation of Allele-Specific Expression in the Presence of Phasing Uncertainty.

Allele-specific expression (ASE) analyses aim to detect imbalanced expression of maternal versus paternal copies of an autosomal gene. Such allelic imbalance can result from a variety of cis-acting causes, including disruptive mutations within one copy of a gene that impact the stability of transcripts, as well as regulatory variants outside the gene that impact transcription initiation. Current methods for ASE estimation suffer from a number of shortcomings, such as relying on only one variant within a gene, assuming perfect phasing information across multiple variants within a gene, or failing to account for alignment biases and possible genotyping errors. We developed BEASTIE, a Bayesian hierarchical model designed for precise ASE quantification at the gene level, based on given genotypes and RNA-Seq data. BEASTIE addresses the complexities of allelic mapping bias, genotyping error, and phasing errors by incorporating empirical phasing error rates derived from Genome-in-a-Bottle individual NA12878. BEASTIE surpasses existing methods in accuracy, especially in scenarios with high phasing errors. This improvement is critical for identifying rare genetic variants often obscured by such errors. Through rigorous validation on simulated data and application to real data from the 1000 Genomes Project, we establish the robustness of BEASTIE. These findings underscore the value of BEASTIE in revealing patterns of ASE across gene sets and pathways. The software is freely available from https://github.com/x811zou/BEASTIE . BEASTIE is available as Python source code and as a Docker image. Additional information is available online.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Allelic bias when performing in-solution enrichment of ancient human DNA.

In-solution hybridisation enrichment of genetic variation is a valuable methodology in human paleogenomics. It allows enrichment of endogenous DNA by targeting genetic markers that are comparable between sequencing libraries. Many studies have used the 1240k reagent-which enriches 1,237,207 genome-wide SNPs-since 2015, though access was restricted. In 2021, Twist Biosciences and Daicel Arbor Biosciences independently released commercial kits that enabled all researchers to perform enrichments for the same 1240 k SNPs. We used the Daicel Arbor Biosciences Prime Plus kit to enrich 132 ancient samples from three continents. We identified a systematic assay bias that increases genetic similarity between enriched samples and that cannot be explained by batch effects. We present the impact of the bias on population genetics inferences (e.g. Principal Components Analysis, ƒ-statistics) and genetic relatedness (READ). We compare the Prime Plus bias to that previously reported of the legacy 1240k enrichment assay. In ƒ-statistics, we find that all Prime-Plus-generated data exhibit artefactual excess shared drift, such that within-continent relationships cannot be correctly determined. The bias is more subtle in READ, though interpretation of the results can still be misleading in specific contexts. We expect the bias may affect analyses we have not yet tested. Our observations support previously reported concerns for the integration of different data types in paleogenomics. We also caution that technological solutions to generate 1240k data necessitate a thorough validation process before their adoption in the paleogenomic community.

P30 A longitudinal perspective of antimicrobial resistance evolution within urinary tract infection-based clinical trials

Abstract Background The mainstay treatments for urinary tract infections (UTIs) are nitrofurantoin and trimethoprim. The common route to acquire trimethoprim resistance (TriR) is via horizontal transfer of the trimethoprim-insensitive dihydrofolate reductase: dfrA. In contrast, nitrofurantoin resistance (NitR) requires the inactivation of two chromosomally encoded nitroreductases: nfsA and nfsB. The difference in antimicrobial resistance (AMR) evolution is reflected in community surveillance data: NitR incidence = ∼10% and for TriR > 30%. Longitudinal clinical UTI trials provide a unique opportunity to monitor in situ evolution of AMR and correlate its appearance to trial outcomes and the impact on uropathogens. Objectives To explore the in situ AMR evolution and its impact in Escherichia coli isolates from patients participating in the clinical trials: AnTIC and ALTAR. AnTIC was an open label randomized trial assessing the efficacy of antibiotic prophylaxis use in clean intermittent self-catheterizing patients. ALTAR compared the efficacy of the non-antibiotic alternative methenamine hippurate to antibiotic prophylaxis to treat recurrent UTIs. Methods The investigation of the evolution of nitrofurantoin and trimethoprim resistance in E. coli used general microbiology techniques, bioinformatic genome analysis and genetics to model known AMR mutations in susceptible E. coli strains. Results Trimethoprim resistance amongst E. coli trial isolates was driven by 14 out of ∼45 known dfrA allelic variants; the most common being dfrA1 and dfrA17. Growth analysis identified an allelic bias when strains were exposed to sub-MIC concentrations of trimethoprim. Growth rate analysis identified a 2%–10% slower doubling time for nitrofurantoin-resistant strains: NitS: 20.8 ± 0.7 min compared to NitR: 23 ± 0.8 min. Statistically, these data suggested no fitness advantage of evolved strains compared to the sensitive predecessor (P value = 0.13). Genetic manipulation of E. coli to mimic NitR evolution, however, supported a selective advantage. Conclusions Longitudinal sampling during antibiotic based clinical trials has provided a new perspective to the response of E. coli to UTI-related antibiotics. Correlation of nitrofurantoin response to pharmacokinetic data suggests that the low incidence of E. coli NitR is driven by selection not fitness. However, fitness correlates to dfrA allele carriage and could potentially be exploited to benefit the use of trimethoprim if other antibiotics are impeded by AMR.

Sequence capture: Obsolete or irreplaceable? A thorough validation across phylogenetic distances and its applicability to hybrids and allopolyploids.

As whole-genome sequencing has become pervasive, some have suggested that reduced genomic representation approaches, for example, sequence capture, are becoming obsolete. In the present study, we argue that these techniques still provide excellent tools in terms of price and quality of data as well as in their ability to provide markers with specific features, as required, for example, in phylogenomics. A potential drawback of the wide-scale application of reduced representation approaches could be their drop in efficiency with increasing phylogenetic distance from the reference species. While some studies have focused on the degree and performance of reduced representation techniques in such situations, to our knowledge, none of them evaluated their applicability to inter-specific hybrids and polyploids. This highlights a significant gap in current knowledge since there is increasing evidence for the frequent occurrence of natural hybrids and polyploids, as well as for the major importance of both phenomena in evolution. The main aim of the present study was to carry out a thorough validation of SEQcap applicability to (1) a set of non-model taxa with a wide range of phylogenetic relatedness and (2) inter-specific hybrids of various ploidies and genomic compositions. Considering the latter point, we especially focused on mechanisms causing allelic bias and consequent allelic dropout, as these could have confounding effects with respect to the evolutionary genomic dynamics of hybrids, especially in asexuals, which virtually reproduce as a frozen F1 generation.

Reassessment of weak parent-of-origin expression bias shows it rarely exists outside of known imprinted regions.

In mouse and human, genes subjected to genomic imprinting have been shown to function in development, behavior, and post-natal adaptations. Failure to correctly imprint genes in human is associated with developmental syndromes, adaptive, and metabolic disorders during life as well as numerous forms of cancer. In recent years researchers have turned to RNA-seq technologies applied to reciprocal hybrid strains of mice to identify novel imprinted genes, causing a threefold increase in genes reported as having a parental origin-specific expression bias. The functional relevance of parental origin-specific expression bias is not fully appreciated especially since many are reported with only minimal parental bias (e.g. 51:49). Here, we present an in-depth meta-analysis of previously generated RNA-seq data and show that the methods used to generate and analyze libraries greatly influence the calling of allele-specific expression. Validation experiments show that most novel genes called with parental-origin-specific allelic bias are artefactual, with the mouse strain contributing a larger effect on expression biases than parental origin. Of the weak novel genes that do validate, most are located at the periphery of known imprinted domains, suggesting they may be affected by local allele- and tissue-specific conformation. Together these findings highlight the need for robust tools, definitions, and validation of putative imprinted genes to provide meaningful information within imprinting databases and to understand the functional and mechanistic implications of the process.

Identification of chilling-tolerant genes in maize via bulked segregant analysis sequencing

Maize (Zea mays L.) is a thermophilic crop that is commonly affected by chilling stress, which adversely affects growth and yield. However, the genetic bases underlying the cold response and acclimation mechanisms remain unclear. Here, we crossed two maize inbred lines that exhibit opposite chilling tolerance at the seedling stage, Dan9046 (D9046; extremely chilling-tolerant) and Jingnuo 6 (JN6; extremely chilling-sensitive). From the 564 F2 individuals evaluated, the 50 most chilling-tolerant and the 50 most chilling-sensitive lines were selected to construct the chilling-tolerant and -sensitive pools, respectively. The two pools and the parental lines were then analyzed via bulked segregant analysis sequencing (BSA-seq). Loci linked to cold tolerance were identified in two regions of chromosome 5 based on single nucleotide polymorphism (SNP)-index and Δ(SNP-index) values. These two genomic intervals contained a total of 70 candidate genes. Of those genes, 49 had homologous genes that previous research has indicated are involved in stress tolerance. Metabolic pathways were enriched according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Between D9046 and JN6, there were a total of 921 SNPs and insertion/deletion mutations (Indels) that either caused a non-synonymous mutation or were located in a non-coding regions. Kompetitive Allele-Specific PCR (KASP) assays revealed that 20 SNPs within six genes exhibited allelic polymorphisms bias among the chilling-tolerant or chilling-sensitive inbred lines, consistent with the BSA-seq data results. Reverse transcription quantitative PCR (RT-qPCR) results revealed differential expression patterns in five of those genes (Zm00001d014321, Zm00001d014324, Zm00001d014325, Zm00001d015212 and Zm00001d015223) between D9046 and JN6 after low-temperature treatment, suggesting that they may have been related to chilling tolerance. In summary, we identified allelic polymorphisms of candidate genes that were related to chilling tolerance, providing genetic resources and KASP markers for future improvement of maize cold tolerance and breeding via marker-assisted selection. Data availabilityData will be made available on request.

Evaluating Stem Cells to Predict Post-Transplant Relapse in the Myelodysplastic Syndromes

Malignant hematopoietic stem cells (HSCs) both initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to frontline non-transplant therapies. Consequently, allogeneic transplant is the only curative therapy, but relapse can still occur. While post-transplant relapse is hypothesized to be driven by persistent MDS HSCs, their detection has proven challenging due to their limiting frequency in biospecimens. The accuracy of sequencing low-input specimens is limited by allelic drop-out and amplification bias. We adapted a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy associated genes that incorporates unique molecular identifiers capable of retaining read depths of >12x from as few as 19 sorted HSCs. We aimed to characterize residual disease after transplant with greater resolution than previously possible by sequencing sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38-CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors. We applied our technique to an MDS case characterized by bi-allelic TP53 mutations (Y220C and P75fs), assessing a bone marrow (BM) specimen D+168 after transplant, which was five months prior to clinical relapse. Both mutations were present at variant allelic frequencies (VAFs) of 8% and 4% in 258 sorted CD34+CD38- cells with 105x coverage (xc), but simultaneously undetectable in bulk BM (481xc) and CD34+CD38+ progenitor cells (239xc) despite greater cell number and read depth. We next evaluated a D+179 post-transplant BM from a case of MDS harboring ASXL1 (R713G), DNMT3A (R882H), and IDH2 (R140Q) mutations. All three mutations were detectable in 102 sorted HSCs/MPPs at VAFs of 20% (92xc), 13% (68xc), and 35% (89xc), respectively, while absent in bulk BM (369xc) and CD34+CD38+ progenitors (506xc). These findings preceded clinical relapse by nearly two years. We additionally investigated pre- and post-transplant BM samples of a patient with a germline DDX41 M1I mutation treated for MDS. In such cases, a second somatic DDX41 mutation is often acquired, impairing ribosomal biogenesis. This mutation is always present at a low VAF, and it is unclear how it promotes disease. Prior to transplant, we identified a somatic DDX41 R525H mutation at a VAF of 2.6% in bulk BM. However, this mutation was present at a VAF of 45% in sorted HSCs/MPPs, revealing the pervasiveness of this "minor clone” at the apex of the hematopoietic hierarchy (Fig. 1). Additionally, two previously unknown TP53 clones were discovered at VAFs of 22% (K132R) and 1.8% (C275F) in HSCs/MPPs, and these were undetectable in bulk BM. At D+102 post-transplant, we detected persistence of both M1I (VAF 43%) and R52H (VAF 27%) DDX41 mutations in 19 sorted HSCs/MPPs (12xc) 78 days prior to relapse. These data suggest that while impaired ribosomal biogenesis in HSCs with bi-allelic DDX41 mutations prevents effective hematopoiesis, it also increases self-renewal and accumulation of HSCs. Next, we sought to test if early identification of residual MDS HSCs post-transplant is predictive of disease relapse. We evaluated a cohort of patients who underwent myeloablative allogeneic transplant for MDS at UTSW and MSKCC (n=21). We evaluated BMs available from post-transplant dates ranging from D+30 to D+120, including any specimen from which we were able to sort at least 15 HSCs/MPPs and achieve an effective read depth of >10. We censored specimens with follow-up of less than one year. With a median follow-up of two years, detection of disease mutations in HSCs/MPPs was 100% specific and 88% sensitive for relapse (Fig. 2). The average time from detection of disease in HSCs to clinical relapse was 6.9 months. Detection in CD38+CD34+ progenitors was also 100% specific but only 67% sensitive for relapse. Assessment of mutations in bulk BM (mean 529xc) was only 22% sensitive and 50% specific for relapse. In conclusion, our research shows for the first time that relapse of MDS after allogeneic transplant is driven by failure to eradicate MDS HSCs, and that detection of MDS HSCs early after transplant is highly predictive for relapse. Validation of this assay in larger cohorts promises to yield a tool to identify patients who may benefit from early post-transplant interventions to forestall relapse. We also demonstrate how this assay can reveal novel insights into human disease HSC biology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Three assays for in-solution enrichment of ancient human DNA at more than a million SNPs.

The strategy of in-solution enrichment for hundreds of thousands of single-nucleotide polymorphisms (SNPs) has been used to analyze >70% of individuals with genome-scale ancient DNA published to date. This approach makes it economical to study ancient samples with low proportions of human DNA and increases the rate of conversion of sampled remains into interpretable data. So far, nearly all such data have been generated using a set of bait sequences targeting about 1.24 million SNPs (the "1240k reagent"), but synthesis of the reagent has been cost-effective for only a few laboratories. In 2021, two companies, Daicel Arbor Biosciences and Twist Bioscience, made available assays that target the same core set of SNPs along with supplementary content. We test all three assays on a common set of 27 ancient DNA libraries and show that all three are effective at enriching many hundreds of thousands of SNPs. For all assays, one round of enrichment produces data that are as useful as two. In our testing, the "Twist Ancient DNA" assay produces the highest coverages, greatest uniformity on targeted positions, and almost no bias toward enriching one allele more than another relative to shotgun sequencing. We also identify hundreds of thousands of targeted SNPs for which there is minimal allelic bias when comparing 1240k data to either shotgun or Twist data. This facilitates coanalysis of the large data sets that have been generated using 1240k and Twist capture, as well as shotgun sequencing approaches.

Variable allelic expression of imprinted genes at the Peg13, Trappc9, Ago2 cluster in single neural cells.

Genomic imprinting is an epigenetic process through which genes are expressed in a parent-of-origin specific manner resulting in mono-allelic or strongly biased expression of one allele. For some genes, imprinted expression may be tissue-specific and reliant on CTCF-influenced enhancer-promoter interactions. The Peg13 imprinting cluster is associated with neurodevelopmental disorders and comprises canonical imprinted genes, which are conserved between mouse and human, as well as brain-specific imprinted genes in mouse. The latter consist of Trappc9, Chrac1 and Ago2, which have a maternal allelic expression bias of ∼75% in brain. Findings of such allelic expression biases on the tissue level raise the question of how they are reflected in individual cells and whether there is variability and mosaicism in allelic expression between individual cells of the tissue. Here we show that Trappc9 and Ago2 are not imprinted in hippocampus-derived neural stem cells (neurospheres), while Peg13 retains its strong bias of paternal allele expression. Upon analysis of single neural stem cells and in vitro differentiated neurons, we find not uniform, but variable states of allelic expression, especially for Trappc9 and Ago2. These ranged from mono-allelic paternal to equal bi-allelic to mono-allelic maternal, including biased bi-allelic transcriptional states. Even Peg13 expression deviated from its expected paternal allele bias in a small number of cells. Although the cell populations consisted of a mosaic of cells with different allelic expression states, as a whole they reflected bulk tissue data. Furthermore, in an attempt to identify potential brain-specific regulatory elements across the Trappc9 locus, we demonstrate tissue-specific and general silencer activities, which might contribute to the regulation of its imprinted expression bias.

Paternal imprinting of dosage-effect defective1 contributes to seed weight xenia in maize

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinted dosage-effect defective1 (ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5–10% seed weight reduction when ded1 is transmitted through the male, while homozygous mutants are defective with a 70–90% seed weight reduction. Ded1 encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

Dynamic molecular evolution of a supergene with suppressed recombination in white-throated sparrows.

In white-throated sparrows, two alternative morphs differing in plumage and behavior segregate with a large chromosomal rearrangement. As with sex chromosomes such as the mammalian Y, the rearranged version of chromosome two (ZAL2m) is in a near-constant state of heterozygosity, offering opportunities to investigate both degenerative and selective processes during the early evolutionary stages of 'supergenes.' Here, we generated, synthesized, and analyzed extensive genome-scale data to better understand the forces shaping the evolution of the ZAL2 and ZAL2m chromosomes in this species. We found that features of ZAL2m are consistent with substantially reduced recombination and low levels of degeneration. We also found evidence that selective sweeps took place both on ZAL2m and its standard counterpart, ZAL2, after the rearrangement event. Signatures of positive selection were associated with allelic bias in gene expression, suggesting that antagonistic selection has operated on gene regulation. Finally, we discovered a region exhibiting long-range haplotypes inside the rearrangement on ZAL2m. These haplotypes appear to have been maintained by balancing selection, retaining genetic diversity within the supergene. Together, our analyses illuminate mechanisms contributing to the evolution of a young chromosomal polymorphism, revealing complex selective processes acting concurrently with genetic degeneration to drive the evolution of supergenes.

IL12RB1 allele bias in human TH cells is regulated by functional SNPs in its 3′UTR

Allele bias is an epigenetic mechanism wherein only the maternal- or paternal-derived allele of a gene is preferentially expressed. Allele bias is used by T cells to regulate expression of numerous genes, including those which govern their development and response to cytokines. Here we demonstrate that human TH cell expression of the cytokine receptor gene IL12RB1 is subject to allele bias, and the extent to which this bias occurs is influenced by cells’ differentiation status and two polymorphic sites in the IL12RB1 3′UTR. The single nucleotide polymorphisms (SNPs) at these sites, rs3746190 and rs404733, function to increase expression of their encoding allele. Modeling suggests this is due to a stabilizing effect of these SNPs on the predicted mRNA secondary structure. The SNP rs3746190 is also proximal to the predicted binding site of microRNA miR-1277, raising the possibility that miR-1277 cannot exert suppression in the presence of rs3746190. Functional experiments demonstrate, however, that miR-1277 suppression of IL12RB1 3′UTR expression—which itself has not been previously reported—is nevertheless independent of rs3746190. Collectively, these data demonstrate that rs3746190 and rs404733 are functional SNPs which regulate IL12RB1 allele bias in human TH cells.

Inference of putative cell-type-specific imprinted regulatory elements and genes during human neuronal differentiation.

Genomic imprinting results in gene expression bias caused by parental chromosome of origin and occurs in genes with important roles during human brain development. However, the cell-type and temporal specificity of imprinting during human neurogenesis is generally unknown. By detecting within-donor allelic biases in chromatin accessibility and gene expression that are unrelated to cross-donor genotype, we inferred imprinting in both primary human neural progenitor cells and their differentiated neuronal progeny from up to 85 donors. We identified 43/20 putatively imprinted regulatory elements (IREs) in neurons/progenitors, and 133/79 putatively imprinted genes in neurons/progenitors. Although 10 IREs and 42 genes were shared between neurons and progenitors, most putative imprinting was only detected within specific cell types. In addition to well-known imprinted genes and their promoters, we inferred novel putative IREs and imprinted genes. Consistent with both DNA methylation-based and H3K27me3-based regulation of imprinted expression, some putative IREs also overlapped with differentially methylated or histone-marked regions. Finally, we identified a progenitor-specific putatively imprinted gene overlapping with copy number variation that is associated with uniparental disomy-like phenotypes. Our results can therefore be useful in interpreting the function of variants identified in future parent-of-origin association studies.

Scelestial: Fast and accurate single-cell lineage tree inference based on a Steiner tree approximation algorithm.

Single-cell genome sequencing provides a highly granular view of biological systems but is affected by high error rates, allelic amplification bias, and uneven genome coverage. This creates a need for data-specific computational methods, for purposes such as for cell lineage tree inference. The objective of cell lineage tree reconstruction is to infer the evolutionary process that generated a set of observed cell genomes. Lineage trees may enable a better understanding of tumor formation and growth, as well as of organ development for healthy body cells. We describe a method, Scelestial, for lineage tree reconstruction from single-cell data, which is based on an approximation algorithm for the Steiner tree problem and is a generalization of the neighbor-joining method. We adapt the algorithm to efficiently select a limited subset of potential sequences as internal nodes, in the presence of missing values, and to minimize cost by lineage tree-based missing value imputation. In a comparison against seven state-of-the-art single-cell lineage tree reconstruction algorithms—BitPhylogeny, OncoNEM, SCITE, SiFit, SASC, SCIPhI, and SiCloneFit—on simulated and real single-cell tumor samples, Scelestial performed best at reconstructing trees in terms of accuracy and run time. Scelestial has been implemented in C++. It is also available as an R package named RScelestial.

Gllac7 Is Induced by Agricultural and Forestry Residues and Exhibits Allelic Expression Bias in Ganoderma lucidum.

Ganoderma lucidum has a wide carbon spectrum, while the expression profile of key genes relevant to carbon metabolism on different carbon sources has been seldom studied. Here, the transcriptomes of G. lucidum mycelia cultured on each of 19 carbon sources were conducted. In comparison with glucose, 16 to 1,006 genes were upregulated and 7 to 1,865 genes were downregulated. Significant gene expression dynamics and induced activity were observed in laccase genes when using agricultural and forestry residues (AFRs) as solo carbon sources. Furthermore, study of laccase gene family in two haploids of G. lucidum GL0102 was conducted. Totally, 15 and 16 laccase genes were identified in GL0102_53 and GL0102_8, respectively, among which 15 pairs were allelic genes. Gene structures were conserved between allelic laccase genes, while sequence variations (most were SNPs) existed. Nine laccase genes rarely expressed on all the tested carbon sources, while the other seven genes showed high expression level on AFRs, especially Gllac2 and Gllac7, which showed 5- to 1,149-fold and 4- to 94-fold upregulation in mycelia cultured for 5 days, respectively. The expression of H53lac7 was consistently higher than that of H8lac7_1 on all the carbon sources except XM, exhibiting a case of allelic expression bias. A total of 47 SNPs and 3 insertions/deletions were observed between promoters of H53lac7 and H8lac7_1, which lead to differences in predicted binding sites of zinc fingers. These results provide scientific data for understanding the gene expression profile and regulatory role on different carbon sources and may support further functional research of laccase.

Assessing homologous recombination deficiency (HRD) in ovarian cancer: Optimizing concordance of the regulatory-approved companion diagnostic and a next-generation sequencing (NGS) assay kit.

e17571 Background: Olaparib + bevacizumab is approved as first-line maintenance treatment of ovarian cancer that is HRD positive, defined as the presence of a deleterious/suspected deleterious BRCA mutation (BRCAm) and/or genomic instability. HRD genomic instability score (GIS) has been assessed in clinical trials using a regulatory-approved companion diagnostic, Myriad myChoice® CDx. We evaluated the performance of an in-development NGS assay kit that identifies BRCA variants based on genomic content from Illumina’s TruSight™ Oncology 500 research assay, and, with additional genomic content, measures HRD GIS in tumor tissue (Illumina test) in parallel. Analytic concordance of the Illumina test versus the Myriad myChoice® PLUS assay (Myriad test) under improved algorithms for calculating GIS scores is reported. Methods: Ovarian cancer tissue samples were analyzed with the Illumina (40 ng DNA; N = 227) and Myriad (200 ng DNA; N = 254) tests. Agreement rates for BRCA analysis, HRD GIS, and overall HRD status (includes BRCAm and HRD GIS [cutoff, 42]) were analyzed. For the overall and the non-BRCAm analysis cohorts, Pearson correlation between the continuous HRD GIS of the Illumina and Myriad tests was determined. Analytic sensitivity and specificity of the Illumina-derived HRD GIS to classify HRD GIS (cutoff, 42) were evaluated using AUROC. After initial concordance calculations, modifications were made to increase the quality of the Illumina preprocessing data (ie, reducing allele dosage noise and allele bias) upstream of the GIS calling algorithm. Reprocessing of the existing raw data led to further improvement in the point estimates of positive and negative percent agreement. Results: Prevalence using the Illumina and Myriad tests was 51.2% and 49.2% (overall HRD positive) and 27.6% and 25.5% (BRCAm), respectively. Agreement rates are reported in the Table. Correlation of the Illumina HRD GIS with Myriad HRD GIS was 0.980 (all samples) and 0.976 (non-BRCAm cohort); AUROCs were 0.995 and 0.992, respectively. Conclusions: Comparison between the Illumina and Myriad tests showed that overall HRD status, BRCA analysis, and HRD GIS detection results were > 90% concordant; HRD GIS was highly correlated (r > 0.98); and prevalence estimates were similar. These data suggest that a distributable kit solution, such as the Illumina test under development, will approach the performance of the Myriad myChoice HRD assay.[Table: see text]

Long-read mapping to repetitive reference sequences using Winnowmap2.

Approximately 5-10% of the human genome remains inaccessible due to the presence of repetitive sequences such as segmental duplications and tandem repeat arrays. We show that existing long-read mappers often yield incorrect alignments and variant calls within long, near-identical repeats, as they remain vulnerable to allelic bias. In the presence of a nonreference allele within a repeat, a read sampled from that region could be mapped to an incorrect repeat copy. To address this limitation, we developed a new long-read mapping method, Winnowmap2, by using minimal confidently alignable substrings. Winnowmap2 computes each read mapping through a collection of confident subalignments. This approach is more tolerant of structural variation and more sensitive to paralog-specific variants within repeats. Our experiments highlight that Winnowmap2 successfully addresses the issue of allelic bias, enabling more accurate downstream variant calls in repetitive sequences.

A cattle graph genome incorporating global breed diversity

Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds, especially those from Africa for which genomic resources are particularly limited, despite their disproportionate importance to the continent’s economies. To mitigate this issue, we have generated assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into a graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1 Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research.

Assessing Bos taurus introgression in the UOA Bos indicus assembly

BackgroundReference genomes are essential in the analysis of genomic data. As the cost of sequencing decreases, multiple reference genomes are being produced within species to alleviate problems such as low mapping accuracy and reference allele bias in variant calling that can be associated with the alignment of divergent samples to a single reference individual. The latest reference sequence adopted by the scientific community for the analysis of cattle data is ARS_UCD1.2, built from the DNA of a Hereford cow (Bos taurus taurus—B. taurus). A complementary genome assembly, UOA_Brahman_1, was recently built to represent the other cattle subspecies (Bos taurus indicus—B. indicus) from a Brahman cow haplotype to further support analysis of B. indicus data. In this study, we aligned the sequence data of 15 B. taurus and B. indicus breeds to each of these references.ResultsThe alignment of B. taurus individuals against UOA_Brahman_1 detected up to five million more single-nucleotide variants (SNVs) compared to that against ARS_UCD1.2. Similarly, the alignment of B. indicus individuals against ARS_UCD1.2 resulted in one and a half million more SNVs than that against UOA_Brahman_1. The number of SNVs with nearly fixed alternative alleles also increased in the alignments with cross-subspecies. Interestingly, the alignment of B. taurus cattle against UOA_Brahman_1 revealed regions with a smaller than expected number of counts of SNVs with nearly fixed alternative alleles. Since B. taurus introgression represents on average 10% of the genome of Brahman cattle, we suggest that these regions comprise taurine DNA as opposed to indicine DNA in the UOA_Brahman_1 reference genome. Principal component and admixture analyses using genotypes inferred from this region support these taurine-introgressed loci. Overall, the flagged taurine segments represent 13.7% of the UOA_Brahman_1 assembly. The genes located within these segments were previously reported to be under positive selection in Brahman cattle, and include functional candidate genes implicated in feed efficiency, development and immunity.ConclusionsWe report a list of taurine segments that are in the UOA_Brahman_1 assembly, which will be useful for the interpretation of interesting genomic features (e.g., signatures of selection, runs of homozygosity, increased mutation rate, etc.) that could appear in future re-sequencing analysis of indicine cattle.