Small Indels Research Articles

BIOM-56. THE PRIVATE AND RECURRING ANTIGENIC PROFILE OF MOST AGGRESIVE PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Efficient immunotherapies for pediatric brain tumors face a significant challenge in identifying tumor rejection antigens. To overcome this hurdle, we utilized a comprehensive computer algorithm called the Open Reading Frame Antigen Network (O.R.A.N.). METHODS Our focus was on expressed epitopes, including single nucleotide variations, small indels, gene fusions, novel junctions from alternative splicing, and Tumor Associated Antigens (TAAs), for antigenic epitope predictions. To ensure uniqueness, we compared immunogenic TAA epitopes against a personalized human proteomic library, ensuring they were not shared by other expressed isoforms or genes. The immunological landscape of medulloblastoma (n = 170) and DIPG (n = 30) was explored using immune deconvolution techniques combined with single-cell RNA sequencing integration. RESULTS Our hypothesis was that medulloblastomas and DIPG exhibit diverse immune profiles and express genes that are rarely found in normal tissues, making them potential targets for immunotherapy. In this study, we found that most neoantigens identified in medulloblastoma patients were unique to each individual, emphasizing the need for personalized neoantigen-based therapies. Interestingly, recurring neoantigens were predominantly present in the MB SHH subgroup, while they constituted only a small fraction of putative antigens in WNT, Group 3, and Group 4 patients. On the other hand, recurring Tumor Associated Antigens (TAAs) were more frequently expressed in the latter three molecular subgroups but not in the SHH subgroup. Thus, a combination of personalized neoantigen vaccines and targeting recurring TAAs could potentially maximize the anti-tumor effects. CONCLUSION Medulloblastoma is a disease characterized by its heterogeneity, and current standard treatments do not adequately address this aspect. In the case of DIPG, surgical resection is not a viable option due to its diffuse and infiltrative nature within the brainstem. By leveraging the power of the immune system, cellular immunotherapy holds promise in providing more targeted and personalized treatment options for pediatric brain tumor patients.

CRISPR/Cas9-mediated mutagenesis of the white gene in an ectoparasitic wasp, Habrobracon hebetor.

The ectoparasitic wasp Habrobracon hebetor (Hymenoptera, Braconidae) can parasitize various species of lepidopteran pests. To maximize its potential for biological control, it is necessary to investigate its gene function through genome engineering. To test the effectiveness of genome engineering system in H. hebetor, we injected the mixture of clustered regularly interspaced short palindromic repeats (CRISPR) -associated (Cas) 9 protein and single guide RNA(s) targeting gene white into embryos. The resulting mutants display a phenotype of eye pigment loss. The phenotype was caused by small indel and is heritable. Then, we compared some biological parameters between wildtype and mutant, and found there were no significant differences in other parameters except for the offspring female rate and adult longevity. In addition, cocoons could be used to extract genomic DNA for genotype during the gene editing process without causing unnecessary harm to H. hebetor. Our results demonstrate that the CRISPR/Cas9 system can be used for H. hebetor genome editing and it does not adversely affect biological parameters of the parasitoid wasps. We also provide a feasible non-invasive genotype detection method using genomic DNA extracted from cocoons. Our study introduces a novel tool and method for studying gene function in H. hebetor, and may contribute to better application of H. hebetor in biocontrol. © 2023 Society of Chemical Industry.

Software pipelines for RNA-Seq, ChIP-Seq and germline variant calling analyses in common workflow language (CWL).

Background: Automating data analysis pipelines is a key requirement to ensure reproducibility of results, especially when dealing with large volumes of data. Here we assembled automated pipelines for the analysis of High-throughput Sequencing (HTS) data originating from RNA-Seq, ChIP-Seq and Germline variant calling experiments. We implemented these workflows in Common workflow language (CWL) and evaluated their performance by: i) reproducing the results of two previously published studies on Chronic Lymphocytic Leukemia (CLL), and ii) analyzing whole genome sequencing data from four Genome in a Bottle Consortium (GIAB) samples, comparing the detected variants against their respective golden standard truth sets. Findings: We demonstrated that CWL-implemented workflows clearly achieved high accuracy in reproducing previously published results, discovering significant biomarkers and detecting germline SNP and small INDEL variants. Conclusion: CWL pipelines are characterized by reproducibility and reusability; combined with containerization, they provide the ability to overcome issues of software incompatibility and laborious configuration requirements. In addition, they are flexible and can be used immediately or adapted to the specific needs of an experiment or study. The CWL-based workflows developed in this study, along with version information for all software tools, are publicly available on GitHub (https://github.com/BiodataAnalysisGroup/CWL_HTS_pipelines) under the MIT License. They are suitable for the analysis of short-read (such as Illumina-based) data and constitute an open resource that can facilitate automation, reproducibility and cross-platform compatibility for standard bioinformatic analyses.

Development and validation of a next-generation sequencing panel for clinical pharmacogenetics

The rapid clinical implementation of next generation sequencing techniques is due to its ability to sequence a large number of genetic regions at lower costs than conventional techniques. However, its use in the field of pharmacogenetics is still very limited. ObjectiveDesign, development, implementation and validation of a clinical pharmacogenetics next-generation sequencing panel. MethodWe developed a panel of hybrid capture probes (SureSelect®) for the analysis of the genetic regions of clinical interest collected by literature search and using Illumina HiSeq 1500® sequencing platform. We developed a bioinformatic algorithm for variant annotation, haplotype inference and determination of structural variants in the genes of interest. The results obtained were validated with Coriell® reference material from the pharmacogenetic repositories. ResultsThe developed panel allows the study of a total of 12,794 regions comprised in 389 genes. Validation results showed a sensitivity greater than 99% for single nucleotide variants and small INDELs. Haplotype imputation was consistent with the consensus results in the characterized reference materials. Furthermore, the developed tool was able to correctly identify different types of CYP2D6 copy number variations as well as a wide variety of HLA-B alleles. ConclusionsThis technology represents an appropriate alternative for its clinical use with advantages over conventional techniques in its throughput and complex gene study capabilities (CYP2D6, HLA-B).

THU209 Small INDELs In The Androgen Receptor Gene: Phenotype Implications And Mechanisms Of Mutagenesis In Androgen Insensitivity Syndrome

Abstract Disclosure: R.L. Batista: None. R.M. Ramos: None. A.F. Afonso: None. R.J. Petroli: None. M.Y. Nishi: None. M.M. Ferrari: None. S. Domenice: None. B.B. Mendonca: None. Small indels are insertions or deletions of one or more nucleotides into the DNA sequence. Indels are highly abundant in human genomes and have contributed massively to genome evolution. However, the role of indels and their underlined mechanisms of mutagenesis in Mendelian disorders still needs to be appreciated. To explore the contribution of indels in a monogenic disorder, we analyzed all indels in the androgen receptor gene, including three novel indels, using Androgen Insensitivity Syndrome (AIS) as a model. The goal was to profile indels in AIS. To build this profile, we analyzed the indel distribution through the AR coding region, compared with genomic and exonic populational data, and analyzed their impact on the AIS phenotype and their likely mechanism of mutagenesis. We present 82 different indels in the AR gene reported in individuals with AIS. In most cases (n=78; 95.1%), the phenotype is complete AIS (CAIS). There are four AR indels reported in mild AIS. Most short indels in the AR gene among AIS are frameshift indels (n=64; 79%). Indels are distributed along the entire AR gene, with NTD domain predominance (n=45; 54.9%), followed by LTD domain (n=28; 34.1%). Most are small deletions (n=49; 59.7%), followed by small insertions (n=29; 35.3%) and complex frameshift indels (n=4; 4.8%). Most indels are short (<10 bp; n=81 = 98.8%), with an average of 2.4 nucleotides by indel. Most indels are at non-repetitive sequences with no changes in copy counting (n = 68; 82.9%). Thirteen indels were identified within tandem repeats (15.8%), whereas seven indels were at homopolymers run (8.5%). Trinucleotide regions (Poly-Q, Poly-G) are prone to indels, but indels also happen in other trinucleotide stretches (Poli-L, Poly-P). Regarding the change in copy counting, thirty-nine (47.5%) indels are CCC indels. Indel reported in MAIS is all located at tandem repeats, while all at non-repetitive sequences caused CAIS. 3.65% of AR indels are at a 6bp consensus sequence (TG A/G A/G G/T A/C). In summary, indels represent a significant portion of the variants identified in the AR gene in patients with AIS. Unlike the non-synonymous variants related to this gene, there is a genotype-phenotype correlation (indel - CAIS congruency). However, small NFS indels at small triplet runs can slightly compromise AR function leading to the mild phenotype. Homopolymer runs, and trinucleotide repeats are exonic regions at risk for indels. Slippage explains half of the cases of AR indels. These results increase the molecular understanding of the androgen receptor disruption and the underlined mechanisms of indel mutagenesis in exonic regions and their implication on phenotype. Presentation: Thursday, June 15, 2023

Hepatocyte nuclear factor 1B deletion, but not intragenic mutation, might be more susceptible to hypomagnesemia.

HNF1B syndrome is caused by defects in the hepatocyte nuclear factor 1B (HNF1B) gene, which leads to maturity-onset diabetes of the young type 5 and congenital organ malformations. This study aimed to identify a gene defect in a patient presenting with diabetes and severe diarrhea, while also analyzing the prevalence of hypomagnesemia and its correlation with the HNF1B genotype. Whole exome sequencing was used to identify responsible point mutations and small indels in the proband and their family members. Multiplex ligation-dependent probe amplification was carried out to identify HNF1B deletions. Furthermore, an analysis of published data on 539 cumulative HNF1B cases, from 29 literature sources, was carried out to determine the correlation between the HNF1B genotype and the phenotype of serum magnesium status. Using multiplex ligation-dependent probe amplification, we identified a de novo heterozygous HNF1B deletion in the patient, who showed dorsal pancreas agenesis and multiple kidney cysts, as detected by magnetic resonance imaging. Magnesium supplementation effectively alleviated the symptoms of diarrhea. Hypomagnesemia was highly prevalent in 192 out of 354 (54.2%) patients with HNF1B syndrome. Compared with patients with intragenic mutations, those with HNF1B deletions were more likely to suffer from hypomagnesemia, with an odds ratio of 3.1 (95% confidence interval 1.8-5.4). Hypomagnesemia is highly prevalent in individuals with HNF1B syndrome, and those with HNF1B deletion are more susceptible to developing hypomagnesemia compared with those with intragenic mutations. The genotype-phenotype associations in HNF1B syndrome have significant implications for endocrinologists in terms of genotype detection, treatment decisions and prognosis assessment.

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation.

Long-read sequencing technologies substantially overcome the limitations of short-reads but have not been considered as a feasible replacement for population-scale projects, being a combination of too expensive, not scalable enough or too error-prone. Here we develop an efficient and scalable wet lab and computational protocol, Napu, for Oxford Nanopore Technologies long-read sequencing that seeks to address those limitations. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the National Institutes of Health Center for Alzheimer's and Related Dementias. Using a single PromethION flow cell, we can detect single nucleotide polymorphisms with F1-score comparable to Illumina short-read sequencing. Small indel calling remains difficult within homopolymers and tandem repeats, but achieves good concordance to Illumina indel calls elsewhere. Further, we can discover structural variants with F1-score on par with state-of-the-art de novo assembly methods. Our protocol phases small and structural variants at megabase scales and produces highly accurate, haplotype-specific methylation calls.

TPIA2: an updated tea plant information archive for Camellia genomics.

The genus Camellia consists of about 200 species, which include many economically important species widely used for making tea, ornamental flowersand edible oil. Here, we present an updated tea plant information archive for Camellia genomics (TPIA2; http://tpia.teaplants.cn) by integrating more novel large-scale genomic, transcriptomic, metabolicand genetic variation datasets as well as a variety of useful tools. Specifically, TPIA2 hosts all currently available and well assembled 10 Camellia genomes and their comprehensive annotations from three major sections of Camellia. A collection of 15 million SNPs and 950950 small indels from large-scale genome resequencing of 350 diverse tea accessions were newly incorporated, followed by the implementation of a novel 'Variation' module to facilitate data retrieval and analysis of the functionally annotated variome. Moreover, 116 Camellia transcriptomes were newly assembled and added, leading to a significant extension of expression profiles of Camellia genes to 13 developmental stages and eight abiotic/biotic treatments. An updated 'Expression' function has also been implemented to provide a comprehensive gene expression atlas for Camellia. Two novel analytic tools (e.g. Gene ID Convert and Population Genetic Analysis) were specifically designed to facilitate the data exchange and population genomics in Camellia. Collectively, TPIA2 provides diverse updated valuable genomic resources and powerful functions, and will continue to be an important gateway for functional genomics and population genetic studies in Camellia.

Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system.

The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V systems typically introducing small indels, and type I system mediating long-range deletions. However, the precision of type I systems for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C system with Cas11c for plant genome editing. The Dvu I-C system was efficient to introduce controllable large fragment deletion up to at least 20 kb using paired crRNAs. The paired-crRNAs design also improved the controllability of deletions for the type I-E system. Dvu I-C system was sensitive to spacer length and mismatch, which was benefit for target specificity. In addition, we showed that the Dvu I-C system was efficient for generating stable transgenic lines in maize and rice with the editing efficiency up to 86.67%. Overall, Dvu I-C system we developed here is powerful for achieving controllable large fragment deletions.

Hundreds of independent midsize deletions mediate DNA loss in wild relatives of Red Jungle Fowl

Small and midsize deletions and insertions (InDels) are major events that play a crucial role in the evolution of genome size and contribute to the genetic and phenotypic diversity of species. In recent years, considerable attention has been given to studying small indels associated with various developmental, growth, and production traits in domestic chicken breeds. Additionally, small and midsize indels have been studied between chicken and phylogenetically more distant species such as duck, turkey, rock pigeon, and other passerine birds. However, the investigation of small and midsize deletions in the wild relatives of chickens has been relatively overlooked until now. To address this gap, our study aimed to identify the presence and distribution of midsize deletions (> 1 Kb) in the wild relatives of chickens. We conducted a comparative genomic analysis using high-quality genomic data from four species belonging to the Gallus genus. Our analysis revealed the existence of >125 midsize deletions in the three other species compared to Gallus gallus (red junglefowl). These midsize deletions were found to be distributed in intergenic regions and within introns of various protein-coding genes but not in the exonic regions of protein-coding genes. Furthermore, we observed a trend between the number of midsize deletions and the phylogenetic distance in the phylogeny of the Gallus genus. The most ancestral species, Gallus varius (green junglefowl), exhibited the highest deletions, followed by Gallus lafayettii (Ceylon junglefowl) and Gallus sonneratii (grey junglefowl). Some protein-coding genes harboring deletions in their introns and upstream regions were associated with body development, production, growth traits, abdominal fat deposition, behavioral patterns such as stress, fear, anxiety, plumage color, and adaptation to extreme climatic conditions. Our study finds that the midsize deletions identified in wild relatives of red junglefowl contribute <1% of DNA loss with a rate of 8–44 Kb/My during the evolution of the Gallus genus.

MAGI-ACMG: Algorithm for the Classification of Variants According to ACMG and ACGS Recommendations.

We have developed MAGI-ACMG, a classification algorithm that allows the classification of sequencing variants (single nucleotide or small indels) according to the recommendations of the American College of Medical Genetics (ACMG) and the Association for Clinical Genomic Science (ACGS). The MAGI-ACMG classification algorithm uses information retrieved through the VarSome Application Programming Interface (API), integrates the AutoPVS1 tool in order to evaluate more precisely the attribution of the PVS1 criterion, and performs the customized assignment of specific criteria. In addition, we propose a sub-classification scheme for variants of uncertain significance (VUS) according to their proximity either towards the "likely pathogenic" or "likely benign" classes. We also conceived a pathogenicity potential criterion (P_POT) as a proxy for segregation criteria that might be added to a VUS after posterior testing, thus allowing it to upgrade its clinical significance in a diagnostic reporting setting. Finally, we have developed a user-friendly web application based on the MAGI-ACMG algorithm, available to geneticists for variant interpretation.

Genome Dynamics and Temperature Adaptation During Experimental Evolution of Obligate Intracellular Bacteria.

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.

Clinical relevance of clonal hematopoiesis and its interference in cell-free DNA profiling of patients with gastric cancer.

Clonal hematopoiesis (CH) is a condition in which healthy individuals have somatic mutations in hematopoietic stem cells. It has been reported with increased risk of hematologic malignancy and cardiovascular disease in the general population, but studies of Korean populations with comorbid disease entities are scarce. White blood cells (WBCs) from patients with gastric cancer (GC) (n=121) were analyzed using a DNA-based targeted (531 genes) panel with customized pipeline designed to detect single nucleotide variants and small indels with low-allele-frequency of≥0.2 %. We defined significant CH variants as having variant allele frequency (VAF)≥2 % among variants found in WBCs. Matched cell-free DNA (cfDNA) samples were also analyzed with the same pipeline to investigate the false-positive results caused by WBC variants in cfDNA profiling. Significant CH variants were detected in 29.8 % ofpatients and were associated with age and male sex. Thenumber of CH variants was associated with a history of anti-cancer therapy and age. DNMT3A and TET2 were recurrently mutated. Overall survival rate of treatment-naïve patients with stage IV GC was higher in those with CH, but Cox regression showed no significant association after adjustment for age, sex, anti-cancer therapy, and smoking history. In addition, we analyzed the potential interference of WBC variants in plasma cell-free DNA testing, which has attracted interest as a complementary method for tissue biopsy. Results showed that 37.0 % (47/127) of plasma specimens harbored at least one WBC variant. VAFs of interfering WBC variants in the plasma and WBC were correlated, and WBC variants with VAF≥4 % in WBC were frequently detected in plasma with the same VAF. This study revealed the clinical impact of CH in Korean patients and suggests the potential for its interference in cfDNA tests.

Identification of grapevine clones via high-throughput amplicon sequencing: a proof-of-concept study.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of four important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivar's reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot, and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

Large-scale rare variant burden testing in Parkinson's disease.

Parkinson's disease has a large heritable component and genome-wide association studies have identified over 90 variants with disease-associated common variants, providing deeper insights into the disease biology. However, there have not been large-scale rare variant analyses for Parkinson's disease. To address this gap, we investigated the rare genetic component of Parkinson's disease at minor allele frequencies <1%, using whole genome and whole exome sequencing data from 7184 Parkinson's disease cases, 6701 proxy cases and 51 650 healthy controls from the Accelerating Medicines Partnership Parkinson's disease (AMP-PD) initiative, the National Institutes of Health, the UK Biobank and Genentech. We performed burden tests meta-analyses on small indels and single nucleotide protein-altering variants, prioritized based on their predicted functional impact. Our work identified several genes reaching exome-wide significance. Two of these genes, GBA1 and LRRK2, have variants that have been previously implicated as risk factors for Parkinson's disease, with some variants in LRRK2 resulting in monogenic forms of the disease. We identify potential novel risk associations for variants in B3GNT3, AUNIP, ADH5, TUBA1B, OR1G1, CAPN10 and TREML1 but were unable to replicate the observed associations across independent datasets. Of these, B3GNT3 and TREML1 could provide new evidence for the role of neuroinflammation in Parkinson's disease. To date, this is the largest analysis of rare genetic variants in Parkinson's disease.

Genomic and Transcriptomic Remodeling by Neoadjuvant Chemoradiotherapy (nCRT) and the Indicative Role of Acquired INDEL Percentage for nCRT Efficacy in Esophageal Squamous Cell Carcinoma

The effect of genomic factors on the response of patients with esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy (nCRT), as well as how nCRT influences the genome and transcriptome of ESCC, remain largely unknown. In total, 137 samples from 57 patients with ESCC undergoing nCRT were collected and subjected to whole-exome sequencing and RNA sequencing analysis. Genetic and clinicopathologic factors were compared between the patients achieving pathologic complete response and patients not achieving pathologic complete response. Genomic and transcriptomic profiles before and after nCRT were analyzed. Codeficiency of the DNA damage repair and HIPPO pathways synergistically sensitized ESCC to nCRT. nCRT induced small INDELs and focal chromosomal loss concurrently. Acquired INDEL% exhibited a decreasing trend with the increase of tumor regression grade (P=.06, Jonckheere's test). Multivariable Cox analysis indicated that higher acquired INDEL% was associated with better survival (adjusted hazard ratio [aHR], 0.93; 95% CI, 0.86-1.01; P=.067 for recurrence-free survival [RFS]; aHR, 0.86; 95% CI, 0.76-0.98; P=.028 for overall survival [OS], with 1% of acquired INDEL% as unit). The prognostic value of acquired INDEL% was confirmed by the Glioma Longitudinal AnalySiS data set (aHR, 0.95; 95% CI, 0.902-0.997; P=.037 for RFS; aHR, 0.96; 95% CI, 0.917-1.004; P=.076 for OS). Additionally, clonal expansion degree was negatively associated with patient survival (aHR, 5.87; 95% CI, 1.10-31.39; P=.038 for RFS; aHR, 9.09; 95% CI, 1.10-75.36; P=.041 for OS, with low clonal expression group as reference) and also negatively correlated with acquired INDEL% (Spearman ρ=-0.45; P=.02). The expression profile was changed after nCRT. The DNA replication gene set was downregulated, while the cell adhesion gene set was upregulated after nCRT. Acquired INDEL% was negatively correlated with the enrichment of the DNA replication gene set (Spearman ρ=-0.56; P=.003) but was positively correlated with the enrichment of the cell adhesion gene set (Spearman ρ=0.40; P=.05) in posttreatment samples. nCRT remodels the genome and transcriptome of ESCC. Acquired INDEL% is a potential biomarker to indicate the effectiveness of nCRT and radiation sensitivity.

Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions.

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.

Characterization of Mammary Tumors Arising from MMTV-PyVT Transgenic Mice.

Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic analyses of mammary tumors arising from MMTV-PyVT mice. To this end, mammary tumors were obtained at 6, 9, 12, and 16 weeks of age for histology and whole-mount analyses. We conducted whole-exome sequencing to identify constitutional and tumor-specific mutations, and genetic variants were identified using the GRCm38/mm10 mouse reference genome. Using hematoxylin and eosin analysis and whole-mount carmine alum staining, we demonstrated the progressive proliferation and invasion of mammary tumors. Frameshift insertions/deletions (indels) were noted in the Muc4. Mammary tumors showed small indels and nonsynonymous single-nucleotide variants but no somatic structural alterations or copy number variations. In summary, we validated MMTV-PyVT transgenic mice as a multistage model for mammary carcinoma development and progression. Our characterization may be used as a reference for guidance in future research.

Diagnostic rate of germline pathogenic variants in pancreatic ductal adenocarcinoma patients using whole genome sequencing.

Identification of germline pathogenic variants in cancer patients is critical for treatment planning, genetic counseling, and health policymaking. However, previous estimates of the prevalence of germline etiology of pancreatic ductal adenocarcinoma (PDAC) were biased because they were based only on sequencing data of protein-coding regions of known PDAC candidate genes. To determine the percentage of patients with PDAC carrying germline pathogenic variants, we enrolled the inpatients from the digestive health clinics, hematology and oncology clinics, and surgical clinics of a single tertiary medical center in Taiwan for whole genome sequencing (WGS) analysis of genomic DNA. The virtual gene panel of 750 genes comprised PDAC candidate genes and those listed in the COSMIC Cancer Gene Census. The genetic variant types under investigation included single nucleotide substitutions, small indels, structural variants, and mobile element insertions (MEIs). In 8 of 24 (33.3%) patients with PDAC, we identified pathogenic/likely pathogenic variants, including single nucleotide substitutions and small indels in ATM, BRCA1, BRCA2, POLQ, SPINK1 and CASP8, as well as structural variants in CDC25C and USP44. We identified additional patients carrying variants that could potentially affect splicing. This cohort study demonstrates that an extensive analysis of the abundant information yielded by the WGS approach can uncover many pathogenic variants that could be missed by traditional panel-based or whole exome sequencing-based approaches. The percentage of patients with PDAC carrying germline variants might be much higher than previously expected.

Comprehensive de novo mutation discovery with HiFi long-read sequencing

BackgroundLong-read sequencing (LRS) techniques have been very successful in identifying structural variants (SVs). However, the high error rate of LRS made the detection of small variants (substitutions and short indels < 20 bp) more challenging. The introduction of PacBio HiFi sequencing makes LRS also suited for detecting small variation. Here we evaluate the ability of HiFi reads to detect de novo mutations (DNMs) of all types, which are technically challenging variant types and a major cause of sporadic, severe, early-onset disease.MethodsWe sequenced the genomes of eight parent–child trios using high coverage PacBio HiFi LRS (~ 30-fold coverage) and Illumina short-read sequencing (SRS) (~ 50-fold coverage). De novo substitutions, small indels, short tandem repeats (STRs) and SVs were called in both datasets and compared to each other to assess the accuracy of HiFi LRS. In addition, we determined the parent-of-origin of the small DNMs using phasing.ResultsWe identified a total of 672 and 859 de novo substitutions/indels, 28 and 126 de novo STRs, and 24 and 1 de novo SVs in LRS and SRS respectively. For the small variants, there was a 92 and 85% concordance between the platforms. For the STRs and SVs, the concordance was 3.6 and 0.8%, and 4 and 100% respectively. We successfully validated 27/54 LRS-unique small variants, of which 11 (41%) were confirmed as true de novo events. For the SRS-unique small variants, we validated 42/133 DNMs and 8 (19%) were confirmed as true de novo event. Validation of 18 LRS-unique de novo STR calls confirmed none of the repeat expansions as true DNM. Confirmation of the 23 LRS-unique SVs was possible for 19 candidate SVs of which 10 (52.6%) were true de novo events. Furthermore, we were able to assign 96% of DNMs to their parental allele with LRS data, as opposed to just 20% with SRS data.ConclusionsHiFi LRS can now produce the most comprehensive variant dataset obtainable by a single technology in a single laboratory, allowing accurate calling of substitutions, indels, STRs and SVs. The accuracy even allows sensitive calling of DNMs on all variant levels, and also allows for phasing, which helps to distinguish true positive from false positive DNMs.