Single Nucleotide Insertion Research Articles

Structures of aberrant spliceosome intermediates on their way to disassembly.

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-electron microscopy structures of post-Bact spliceosome intermediates from Schizosaccharomyces pombe primed for disassembly. We identify the DEAH-box helicase-G-patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an overstabilization of the U5 loop I interaction with the 5' exon leading to a single-nucleotide insertion at the 5' splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.

Genetic variations suggests that Takifugu rubripes, T. chinensis, and T. pseudommus are the same species with a shared gene pool

IntroductionPufferfish are valuable ingredients and important fishery resources in Asia. Among them, approximately 25 Takifugu species have undergone explosive speciation in the marine environments of East Asia. Specifically, T. rubripes, T. pseudommus, and T. chinensis are remarkably similar in appearance and genetics, and their classification remains controversial. Here, we provide comprehensive genetic and genomic evidence regarding their speciation and classification.MethodsWhole genome sequencing was performed on T. pseudommus, leading to the identification and development of 15 novel tandem simple sequence repeats (SSRs) from its genomic data. Genetic diversity, differentiation, and population structure among T. rubripes, T. pseudommus, T. chinensis, and T. xanthopterus were subsequently analyzed using a combination of these 15 SSR loci and mitochondrial cytochrome oxidase subunit I (CoI) and cytochrome b (CytB) gene sequences. Furthermore, genomic variations, including single nucleotide polymorphisms, insertions, and deletions, were identified by comparing the genome of T. pseudommus with the reference T. rubripes genome retrieved from the National Center for Biotechnology Information. These variations were screened using ENSEMBL annotation and gene ontology analysis, and their potential association with morphological differences, such as dorsal spot patterns, was evaluated using reference Takifugu specimens.Results and discussionPopulation genetic analysis of T. rubripes, T. pseudommus, and T. chinensis using the CoI and CytB genes, combined with the 15 SSR loci, revealed clustering into a single genetic group characterized by remarkably low genetic diversity (four haplotypes, with diversity values ranging from 0.0000 to 0.00065) and minimal pairwise differentiation (microsatellite-based FST values ranging from -0.0021 to 0.0075). Additionally, comparative genomic analysis between T. pseudommus and the reference T. rubripes genome did not identify genetic variations that could directly explain the observed morphological differences among the species. These findings strongly suggest that T. rubripes, T. pseudommus, and T. chinensis represent a single species sharing a common genetic pool.

A genome-wide association study implicates the olfactory system in Drosophila melanogaster diapause-associated lifespan extension and fecundity.

The effects of environmental stress on animal life are gaining importance with climate change. Diapause is a dormancy program that occurs in response to an adverse environment, followed by resumption of development and reproduction upon the return of favorable conditions. Diapause is a complex trait, so we leveraged the Drosophila genetic reference panel (DGRP) lines and conducted a Genome-Wide Association Study (GWAS) to characterize the genetic basis of diapause. We assessed post-diapause and non-diapause fecundity across 193 DGRP lines. GWAS revealed 546 genetic variants, encompassing single nucleotide polymorphisms, insertions and deletions associated with post-diapause fecundity. We identified 291 candidate diapause-associated genes, 40 of which had previously been associated with diapause, and 89 of which were associated with more than one SNP. Gene network analysis indicated that the diapause-associated genes were primarily linked to neuronal and reproductive system development. Similarly, comparison with results from other fly GWAS revealed the greatest overlap with olfactory-behavior-associated and fecundity-and-lifespan-associated genes. An RNAi screen of selected candidates identified two neuronal genes, Dip-𝛾 and Scribbler, to be required during recovery for post-diapause fecundity. We complemented the genetic analysis with a test of which neurons are required for successful diapause. We found that although amputation of the antenna had little to no effect on non-diapause lifespan, it reduced diapause lifespan and postdiapause fecundity. We further show that olfactory receptor neurons and temperature-sensing neurons are required for successful recovery from diapause. Our results provide insights into the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila .

P59 Clinic and genetic characteristics of patients with generalized pustular psoriasis: a bidirectional cohort study

Abstract Generalized pustular psoriasis (GPP) is an infrequent disease with significant genetic predisposition, the IL36RN gene has been evidenced of pathogenicity, additionally, furthermore, eight genes including CARD14, AP1S3, MPO, SERPINA1, SERPINA3, BTN3A3, TNIP1 and TGFBR2 have been successively identified of involved in the pathogenesis of GPP.1,2 However, a considerable proportion of quintessential GPP patients either exhibit no mutations in the aforementioned genes, or been detected of only single-allele heterozygous variation, or have found two or more pathogenic genes. The prevailing view thought GPP follows an oligogenic inheritance pattern rather than a monogenic one. Currently, genetic testing for GPP employs methodologies such as whole-exome sequencing, next-generation sequencing (NGS) targeted gene panel exome sequencing and Sanger sequencing for specific genes like IL36RN. However, the aforementioned methods do not address the identification of unknown genes, variations in non-coding regions, structural variants (SVs), or copy number variations (CNVs). Whole-genome sequencing (WGS), by comparing individual genomic sequences to a reference genome, provides detailed information on single nucleotide variations (SNVs), insertions and deletions (InDels), SVs and CNVs within the genome. The application of WGS to the research of GPP will undoubtedly contribute to the investigation of its unknown mechanisms. In our study, WGS was carried out on a total of 79 patients diagnosed with pustular psoriasis (PP) up to the present, comprising single GPP (35), psoriasis vulgaris (PsV)-GPP (15), and palmoplantar pustulosis (PPP) or acrodermatitis continua (ACH)-GPP (4), single PPP (10) or ACH (2), clinic characteristics including groove or map-like tongue, nail involvements were also recorded for phenotypic matching. Four variant types (c.115+6T>C, c.334G>A, c.227C>T and c.169G>A) of IL36RN in 43 patients (43/79) were identified, among which c.115 + 6T>C exhibited the highest frequency. Among them, 19 patients had c.115 + 6T>C homozygous mutations, 15 patients had c.115 + 6T>C single heterozygous mutation and 2 patients had c.115 + 6T>C and c.227C>T complex heterozygous mutations. A total of 21/43 patients had IL36RN biallelic mutations, and 22/43 patients had IL36RN monoallelic mutations. In addition, some predicted pathogenic mutations were detected in TNIP1, SERPINA1, MPO, TGFBR2, AP1S3 and CARD14 genes.

GWAS analysis for plant height and stem diameter in sorghum using multiple phenotyping approaches

AbstractSorghum (Sorghum bicolor (L.) Moench) is an important cereal crop cultivated around the globe. To identify the genomic regions responsible for plant height and stem diameter, a subset of the sorghum association panel was phenotyped using manual, ground‐robot, and drone‐based phenotyping approaches during 2019 and 2020 at Tifton, GA. Manual and ground‐robot‐based plant heights had a lower correlation (r = 0.55) than the manual and drone‐based measurements (r = 0.61). Tukey's mean difference plot analysis indicated that both high‐throughput methods were less accurate with taller accessions. In genome‐wide association studies using single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and copy number variants, a total of 136 and 18 SNP loci associated with plant height were identified using mixed linear model and fixed and random model circulating probability unification models. For the first time, we report eight significant SNPs associated with stem diameter using a ground‐based robot. We were able to detect all four major dwarfing genes (Dw1 through Dw4) using manual phenotyping; in contrast, Dw2 was identified using both manual and ground‐robot‐based phenotyping. Sorbi.3004G093400, a gene located 19 kb upstream of the SNP locus SBI‐04_7987371 and a member of the glycoside hydrolase superfamily 35, plays a role in stem diameter, plant height, and is involved in cellular processes. Novel associations (eight for stem diameter), SNPs, and structural variants identified in this study can be used for manipulating plant height and stem diameter in sorghum.

Accumulation of Anthocyanin in the Aleurone of Barley Grains by Targeted Restoration of the MYC2 Gene.

Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented barley, a single nucleotide insertion in this gene causes a frameshift mutation with a premature stop codon. It was hypothesized that restoring the MYC2 reading frame could activate anthocyanin accumulation in the aleurone. Using a targeted mutagenesis approach in the present study, the reading frame of MYC2 was restored in the non-pigmented cultivar Golden Promise. Genetic constructs harboring cas9 and gRNA expression units were developed, pre-validated in protoplasts, and then functional MYC2 alleles were generated at the plant level via Agrobacterium-mediated transformation. Anthocyanin accumulation in the aleurone layer of grains from these mutants was confirmed through microscopy and chemical analysis. The expression of anthocyanin biosynthesis genes was analyzed, revealing that the restoration of MYC2 led to increased transcript levels of F3H and ANS genes. These results confirm the critical role of the MYC2 transcription factor in the blue aleurone trait and provide a biotechnological solution for enriching barley grain with anthocyanins.

In vitro slow-growth conservation, acclimatization, and genetic stability of virus-free apple plants

In vitro slow-growth storage has long played an important role in maintaining valuable horticultural materials. It is particularly applicable to the conservation of virus-free materials recovered from meristem culture or shoot-tip cryotherapy. In this study, the apple cultivar ‘Yanfu-6’ and the rootstock genotype ‘Qingzhen-1’ obtained from a virus disinfection program were compared during the establishment of in vitro slow-growth storage programs. At room temperature (25℃), combining with 4.5% sucrose or 0.5% mannitol, extended the conservation period of ‘Yanfu-6’ and ‘Qingzhen-1’to 5 and 9 months, respectively. Decreasing the temperature to 12℃ led to further reduced shoot growth, extending the conservation period to 9 months for ‘Yanfu-6’, while more than 80% of ‘Qingzhen-1’ shoots could be recovered after one year of storage. Similarly, high rooting and acclimatization success levels were obtained for ‘Qingzhen-1’ after one-year storage at 12℃, as well as for the plants that underwent monthly subcultures, but not for ‘Yanfu-6’. The inability to root in ‘Yanfu-6’ was overcome by micrografting onto rootstock ‘Qingzhen-1’, which resulted in a rooting percentage of 83% and an acclimatization success of 77%. In the analysis of genetic stability by next-generation sequencing, reduced levels of single nucleotide polymorphism (SNPs) and insertions and deletions (InDels) were detected in ‘Qingzhen-1’ shoots recovered after one-year storage at 12℃, as compared with shoots that underwent regular subcultures. These results highlight the use of in vitro slow-growth program assisted with micrografting for the conservation of valuable horticultural species.

Establishment of an efficient and wide-spectrum droplet-vitrification cryopreservation for raspberry (Rubus idaeus L.) germplasm and assessments of genetic integrity and vegetative growth in the regenerants

By optimizing size of shoot tips, preculture medium and exposure duration to PVS2, we established an efficient and wide-spectrum droplet-vitrification cryopreservation for shoot tips of raspberry (Rubus idaeus L.). This protocol yielded 80–100% and 67–100% of survival and shoot regrowth levels in cryopreserved shoot tips across 23 raspberry genotypes. Genetic integrity was assessed in cryo-derived regenerants after 3 months of post-cryopreservation using inter simple sequence repeat (ISSR), single nucleotide polymorphism (SNPs), and insertions and deletions (InDels). ISSR did not detect any polymorphic bands in the cryo-derived regenerants. Although the number of SNPs and InDels decreased in the cryo-derived regenerants, variation trends were similar between the cryo-derived regenerants and the control. Plant vegetative growth and root growth were assessed in the cryo-derived plants after 9 weeks of growth in greenhouse. There were no significant differences in plant vegetative growth measured by plant height, number of fully-opened leaved, leaf area, and fresh and dry weight between the cryo-derived plants and the control, although significant differences were observed in root growth measured by root total length, root average diameter and root volume between the two types of plants. The results obtained in the present study indicate that the droplet-vitrification method has great potential for cryopreservation of raspberry germplasm.

Mutational landscape induced by chronic exposure to environmental PM10 and PM2.5 in A549 lung epithelial cell.

Exposure to particulate matter (PM) has been linked to an increased risk of multiple diseases, primarily lung cancer, through various molecular mechanisms. However, the mutagenic potential of PM remains unclear. This study aimed to provide a comprehensive description of genetic mutations and mutagenic signatures resulting from chronic exposure to PM10 or PM2.5. Using whole exome sequencing, we identified driver mutations and mutational signatures in A549cells, a lung epithelial cell model subjected to weekly exposure to either PM10 or PM2.5, for a period of 28 weeks. The number of single nucleotide variations, insertions, and deletions increased depending on the duration of exposure. PM10 generated the highest number of genomic alterations. Amplifications in SYK (oncogene) and mutations in NCOR1 (tumor suppressor gene) were prevalent in cells exposed to either PM10 or PM2.5; however, other mutations were exclusive, such as TP53 and ANK3 for PM10, and ERCC1 and ERCC2 for PM2.5. Different p53-related signaling pathways were most enriched by driver mutations upon exposure to both PM10 and PM2.5, particularly the glucose deprivation pathway. Exposure to either PM10 or PM2.5 resulted in high frequencies of C>A substitutions and one-base insertions/deletions in microhomology sites. The single-base substitution (SBS) signature SBS05, related to the nucleotide excision DNA repair pathway, contributed the most to both PM10-and PM2.5-exposed cells. The contribution of signature SBS18, related to oxidative stress, was observed in cells exposed to either PM10 or PM2.5, but a greater contribution was observed in PM2.5-exposed cells. In addition, SBS03 and SBS36, which are related to different DNA damage repair mechanisms, were observed more frequently in PM10-exposed cells. We assessed the mutagenic potential of PM10 and PM2.5, as a complete mixture, identifying mutated driver genes and mutational signatures generated by chronic PM exposure, which could contribute to the development of cancer, cardiovascular, and digestive diseases.

Population Genetic Characteristics of the Cultivated Coffea arabica with Whole-Genome Resequencing

Coffea arabica L. (C. arabica) is an economically important agricultural crop and the most popular beverage worldwide. To analyze genetic diversity and provide genetic resources for the selection and breeding of superior varieties of C. arabica, 61 cultivated Arabica coffee accessions were analyzed in the study, including 12 resequencing accessions from previous research and 49 accessions that were resequenced in this study. Single nucleotide polymorphisms (SNPs) and insertion–deletions (InDels) were statistically analyzed. Based on SNP variations, a genetic structure analysis, phylogenetic tree construction, and principal component analysis were performed for the 61 coffee accessions. The results showed that a total of 805.46 Gb of raw whole-genome resequencing data was obtained from the 61 coffee accessions, with 781.29 Gb of high-quality sequencing data after filtering. In total, 7,013,820 SNP sites and 1,074,329 InDel sites were detected. The average sequencing depth ranged from 6.69× to 19.35×, and the coverage ranged from 85.49% to 96.43%. The population genetic structure and phylogenetic analysis of the 61 coffee accessions revealed four lineages, suggesting that they had at least four ancestral genetic components. Catimor exhibited the highest genetic diversity, while Geisha had the lowest genetic diversity. The selective sweep analysis indicated that among the selected genes in Catimor, disease-resistance genes were significantly more numerous than in other coffee varieties. The genome resequencing data and genetic markers identified from the 61 cultivated Arabica coffee materials provided insights into the genetic variation in Arabica coffee germplasm and facilitated extensive genetic research.

Comprehensive genome analysis and variant detection at scale using DRAGEN.

Research and medical genomics require comprehensive, scalable methods for the discovery of novel disease targets, evolutionary drivers and genetic markers with clinical significance. This necessitates a framework to identify all types of variants independent of their size or location. Here we present DRAGEN, which uses multigenome mapping with pangenome references, hardware acceleration and machine learning-based variant detection to provide insights into individual genomes, with ~30 min of computation time from raw reads to variant detection. DRAGEN outperforms current state-of-the-art methods in speed and accuracy across all variant types (single-nucleotide variations, insertions or deletions, short tandem repeats, structural variations and copy number variations) and incorporates specialized methods for analysis of medically relevant genes. We demonstrate the performance of DRAGEN across 3,202 whole-genome sequencing datasets by generating fully genotyped multisample variant call format files and demonstrate its scalability, accuracy and innovation to further advance the integration of comprehensive genomics. Overall, DRAGEN marks a major milestone in sequencing data analysis and will provide insights across various diseases, including Mendelian and rare diseases, with a highly comprehensive and scalable platform.

A single-nucleotide insertion in Rxp confers durable resistance to bacterial pustule in soybean.

The soybean Rxp gene, encoding a bHLH transcription factor and an ACT-like domain, has an rxp allele producing a truncated protein that confers resistance to pustule-causing Xanthomonas axonopodis pv. glycines. In soybean, bacterial pustules caused by Xanthomonas axonopodis pv. glycines lead to premature defoliation and decreased yield in warm, wet climates. In the USA, approximately 70years ago, bacterial pustules were eliminated by introducing a recessive resistance allele, rxp, of the Rxp gene, representing the first example of successful soybean breeding for durable disease resistance in North America. In this study, we isolated this historical Rxp gene from resistant soybean varieties using positional cloning. The 1.06Mb region where Rxp was reported to reside was narrowed down to an 11.1kb region containing a single gene, Glyma.17g090500. The resistance allele, rxp, contains a T insertion. A complementation test of the Rxp allele in resistant plants confirmed the identification of the Rxp gene. The product of the susceptible wild-type allele, Rxp, is presumed to be a basic helix-loop-helix (bHLH) transcription factor with an aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain. This gene was mainly expressed in extended leaves, and its homologs were identified to be distributed in angiosperms. A total of six alleles were obtained: four from spontaneous variation, including the wild-type and three mutant alleles that encoded truncated proteins, and two from ethyl methanesulfonate mutants, including an allele that encoded a truncated protein and a missense allele. By evaluating the resistance of these six alleles, we found that the loss of function of RXP decreased the bacterial pustule lesions. This study provides important insights into the soybean rxp allele, which confers durable resistance to bacterial pustules.

Discovering Novel Proteoforms Using Proteogenomic Workflows Within the Galaxy Bioinformatics Platform.

Proteogenomics is a growing "multi-omics" research area that combines mass spectrometry-based proteomics and high-throughput nucleotide sequencing technologies. Proteogenomics has helped in genomic annotation for organisms whose complete genome sequences became available by using high-throughput DNA sequencing technologies. Apart from genome annotation, this multi-omics approach has also helped researchers confirm expression of variant proteins belonging to unique proteoforms that could have resulted from single-nucleotide polymorphism (SNP), insertion and deletions (Indels), splice isoforms, or other genome or transcriptome variations.A proteogenomic study depends on a multistep informatics workflow, requiring different software at each step. These integrated steps include creating an appropriate protein sequence database, matching spectral data against these sequences, and finally identifying peptide sequences corresponding to novel proteoforms followed by variant classification and functional analysis. The disparate software required for a proteogenomic study is difficult for most researchers to access and use, especially those lacking computational expertise. Furthermore, using them disjointedly can be error-prone as it requires setting up individual parameters for each software. Consequently, reproducibility suffers. Managing output files from each software is an additional challenge. One solution for these challenges in proteogenomics is the open-source Web-based computational platform Galaxy. Its capability to create and manage workflows comprised of disparate software while recording and saving all important parameters promotes both usability and reproducibility. Here, we describe a workflow that can perform proteogenomic analysis on a Galaxy-based platform. This Galaxy workflow facilitates matching of spectral data with a customized protein sequence database, identifying novel protein variants, assessing quality of results, and classifying variants along with visualization against the genome.

Genome-Wide Identification and Expression Analysis of the CLAVATA3/ESR-Related Gene Family in Tiger Nut

CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-related (CLE) genes encode a group of peptide hormones, which coordinate cell proliferation and differentiation in plants. Tiger nut (Cyperus esculentus L.) is a perennial monocot plant that produces oil-rich tubers. However, the mechanisms regulating tuber development in tiger nut are poorly understood, and nothing is known about CLE genes in tiger nut. In this study, we identified 34 CLE genes in the genomes, proteomes, and transcriptomes of C. esculentus (CeCLE). We analyzed their gene structures and expression profiles in different parts of the plant, at three stages of tuber development and in roots in response to dehydration stress. We found a relatively high expression level of CeCLE13 in growing tuber and suggested that the corresponding CLE peptide could be involved in the regulation of tuberization. We also analyzed CeCLE gene sequences in the genome of the most productive K-17 variety in the N. I. Vavilov All-Russian Institute of Plant Genetic Resources collection and found many single nucleotide polymorphisms, insertions, and deletions. Our data provides fundamental information for future research on tiger nut growth and tuberization.

SAKit: An all-in-one analysis pipeline for identifying novel proteins resulting from variant events at both large and small scales.

Background: Genetic mutations that cause the inactivation or aberrant activation of essential proteins may trigger alterations or even dysfunctions in cellular signaling pathways, culminating in the development of precancerous lesions and cancer. Mutations and such dysfunctions can result in the generation of "novel proteins" that are not part of the conventional human proteome. Identification of these proteins carries a profound potential for unraveling promising drug targets and designing innovative therapeutic models. Despite the emergence of diverse tools for detecting DNA or RNA variants, facilitated by the widespread adoption of nucleotide sequencing technology, these methods primarily target point mutations and exhibit suboptimal performance in detecting large-scale and combinatorial mutations. Additionally, the outcomes of these tools are confined to the genome and transcriptome levels, and do not provide the corresponding protein information resulting from genetic alterations. Results: We present the development of Sequencing Analysis Kit (SAKit), a bioinformatics pipeline for hybrid sequencing analysis integrating long-read and short-read RNA sequencing data. Long reads are utilized for detecting large-scale variations such as gene fusions, exon skipping, intron retention, and aberrant expression in non-coding regions, owing to their excellent coverage capabilities. Short reads serve to validate these findings at breakpoints and splice junctions. Conversely, short reads are employed for identifying small-scale variations, including single nucleotide variants, deletions, and insertions, due to their superior sequencing depth, with long reads providing additional validation. SAKit is designed to perform analyses using inter-species configuration files comprising genome references and annotation data, making it applicable to both human and mouse studies. Furthermore, SAKit implements a hierarchical filtering approach to eliminate low-confidence variants and employs open reading frame (ORF) analysis to translate identified variants into protein sequences. Conclusion: SAKit is a robust and versatile bioinformatics tool designed for the comprehensive identification of both large-scale and small-scale variants from RNA-seq data, facilitating the discovery of novel proteins. This pipeline integrates analysis of long-read and short-read sequencing data, offering a powerful solution for researchers in genomics and transcriptomics. SAKit is freely accessible and open-source, available through GitHub (https://github.com/therarna/SAKit) and as a Docker image https://hub.docker.com/repository/docker/therarna). Implemented primarily within a Snakemake framework using Python, SAKit ensures reproducibility, scalability, and ease of use for the scientific community.

Precision Genome Editing Techniques in Gene Therapy: Current State and Future Prospects.

Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPRCas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.

ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.

Genetic evolution analysis of Chinese bayberry germplasm resources in Southern Zhejiang with single nucleotide polymorphism (SNP) and insertion deletion (InDel) markers

Genetic evolution analysis of Chinese bayberry germplasm resources in Southern Zhejiang with single nucleotide polymorphism (SNP) and insertion deletion (InDel) markers

Genomic landscape of early-stage prostate adenocarcinoma in Mexican patients: an exploratory study

BackgroundHealth disparities have been highlighted among patient with prostate adenocarcinoma (PRAD) due to ethnicity. Mexican men present a more aggressive disease than other patients resulting in less favorable treatment outcome. We aimed to identify the mutational landscape which could help to reduce the health disparities among minority groups and generate the first genomics exploratory study of PRAD in Mexican patients.MethodsParaffin-embedded formalin-fixed tumoral tissue from 20 Mexican patients with early-stage PRAD treated at The Instituto Nacional de Cancerología, Mexico City from 2017 to 2019 were analyzed. Tumoral DNA was prepared for whole exome sequencing, the resulting files were mapped against h19 using BWA-MEM. Strelka2 and Lancet packages were used to identify single nucleotide variants (SNV) and insertions or deletions. FACETS was used to determine somatic copy number alterations (SCNA). Cancer Genome Interpreter web interface was used to determine the clinical relevance of variants.ResultsPatients were in an early clinical stage and had a mean age of 59.55 years (standard deviation [SD]: 7.1 years) with 90% of them having a Gleason Score of 7. Follow-up time was 48.50 months (SD: 32.77) with recurrences and progression in 30% and 15% of the patients, respectively. NUP98 (20%), CSMD3 (15%) and FAT1 (15%) were the genes most frequently affected by SNV; ARAF (75%) and ZNF419 (70%) were the most frequently affected by losses and gains SNCA’s. One quarter of the patients had mutations useful as biomarkers for the use of PARP inhibitors, they comprise mutations in BRCA, RAD54L and ATM. SBS05, DBS03 and ID08 were the most common mutational signatures present in this cohort. No associations with recurrence or progression were identified.ConclusionsThis pilot study reveals the mutational landscape of early-stage prostate adenocarcinoma in Mexican men, providing a first approach to understand the mutational patterns and actionable mutations in early prostate cancer can inform personalized treatment approaches and reduce the underrepresentation in genomic cancer studies.

SARS-CoV-2 Omicron subvariant genomic variation associations with immune evasion in Northern California: A retrospective cohort study.

The possibility of association between SARS-CoV-2 genomic variation and immune evasion is not known among persons with Omicron variant SARS-CoV-2 infection. In a retrospective cohort, using Poisson regression adjusting for sociodemographic variables and month of infection, we examined associations between individual non-lineage defining mutations and SARS-CoV-2 immunity status, defined as a) no prior recorded infection, b) not vaccinated but with at least one prior recorded infection, c) complete primary series vaccination, and/or d) primary series vaccination and ≥ 1 booster. We identified all non-synonymous single nucleotide polymorphisms (SNPs), insertions and deletions in SARS-CoV-2 genomes with ≥5% allelic frequency and population frequency of ≥5% and ≤95%. We also examined correlations between the presence of SNPs with each other, with subvariants, and over time. Seventy-nine mutations met inclusion criteria. Among 15,566 persons infected with Omicron SARS-CoV-2, 1,825 (12%) were unvaccinated with no prior recorded infection, 360 (2%) were unvaccinated with a recorded prior infection, 13,381 (86%) had a complete primary series vaccination, and 9,172 (58%) had at least one booster. After examining correlation between SNPs, 79 individual non-lineage defining mutations were organized into 38 groups. After correction for multiple testing, no individual SNPs or SNP groups were significantly associated with immunity status levels. Genomic variation identified within SARS-CoV-2 Omicron specimens was not significantly associated with immunity status, suggesting that contribution of non-lineage defining SNPs to immune evasion is minimal. Larger-scale surveillance of SARS-CoV-2 genomes linked with clinical data can help provide information to inform future vaccine development.