Combining Next-generation Sequencing Research Articles

Development of a Human B7-H3-Specific Antibody with Activity against Colorectal Cancer Cells through a Synthetic Nanobody Library.

Nanobodies have emerged as promising tools in biomedicine due to their single-chain structure and inherent stability. They generally have convex paratopes, which potentially prefer different epitope sites in an antigen compared to traditional antibodies. In this study, a synthetic phage display nanobody library was constructed and used to identify nanobodies targeting a tumor-associated antigen, the human B7-H3 protein. Combining next-generation sequencing and single-clone validation, two nanobodies were identified to specifically bind B7-H3 with medium nanomolar affinities. Further characterization revealed that these two clones targeted a different epitope compared to known B7-H3-specific antibodies, which have been explored in clinical trials. Furthermore, one of the clones, dubbed as A6, exhibited potent antibody-dependent cell-mediated cytotoxicity (ADCC) against a colorectal cancer cell line with an EC50 of 0.67 nM, upon conversion to an Fc-enhanced IgG format. These findings underscore a cost-effective strategy that bypasses the lengthy immunization process, offering potential rapid access to nanobodies targeting unexplored antigenic sites.

Autosomal dominant chronic tubulointerstitial nephropathy: do not forget amyloidosis

Amyloidosis is a rare cause of inherited kidney disease, with most variants responsible for prominent glomerular involvement. In this issue, Kmochová etal. reported the first description of autosomal dominant medullary amyloidosis due to apolipoprotein A4 variants, resulting in slowly progressive chronic kidney disease with minimal proteinuria. Combining next-generation sequencing with histopathological studies incorporating Congo red staining and mass spectrometry should be considered in the diagnostic workup of hereditary tubulointerstitial disorders not identified after routine genetic testing.

Complete genome sequence of a novel robigovirus infecting Mentha arvensis.

The complete genomic sequence of a novel robigovirus, provisionally named "Mentha arvensis robigovirus 1" (MARV1), was determined by combining next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE) PCR. The complete genomic sequence of this new virus is 7617 nucleotides in length, excluding the 3' poly(A) tail. The MARV1 genome encodes a putative replicase, "triple gene block" proteins, and a coat protein. Phylogenetic analysis demonstrated that MARV1 is a member of the genus Robigovirus, with closest relationships to African oil palm ringspot virus (AOPRV). Furthermore, MARV1-derived small interfering RNAs (siRNAs) showed typical patterns of plant-virus-derived siRNAs produced by the host antiviral RNA interference pathway. This is the first report of a plant virus of the genus Robigovirus in M. arvensis.

In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening

The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gβγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo. This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.

RGT: a toolbox for the integrative analysis of high throughput regulatory genomics data

BackgroundMassive amounts of data are produced by combining next-generation sequencing with complex biochemistry techniques to characterize regulatory genomics profiles, such as protein–DNA interaction and chromatin accessibility. Interpretation of such high-throughput data typically requires different computation methods. However, existing tools are usually developed for a specific task, which makes it challenging to analyze the data in an integrative manner.ResultsWe here describe the Regulatory Genomics Toolbox (RGT), a computational library for the integrative analysis of regulatory genomics data. RGT provides different functionalities to handle genomic signals and regions. Based on that, we developed several tools to perform distinct downstream analyses, including the prediction of transcription factor binding sites using ATAC-seq data, identification of differential peaks from ChIP-seq data, and detection of triple helix mediated RNA and DNA interactions, visualization, and finding an association between distinct regulatory factors.ConclusionWe present here RGT; a framework to facilitate the customization of computational methods to analyze genomic data for specific regulatory genomics problems. RGT is a comprehensive and flexible Python package for analyzing high throughput regulatory genomics data and is available at: https://github.com/CostaLab/reg-gen. The documentation is available at: https://reg-gen.readthedocs.io

Acute Transcriptomic and Epigenetic Alterations at T12 After Rat T10 Spinal Cord Contusive Injury.

Spinal cord injury is a severely debilitating condition affecting a significant population in the USA. Spinal cord injury patients often have increased risk of developing persistent neuropathic pain and other neurodegenerative conditions beyond the primary lesion center later in their life. The molecular mechanism conferring to the "latent" damages at distal tissues, however, remains elusive. Here, we studied molecular changes conferring abnormal functionality at distal spinal cord (T12) beyond the lesion center (T10) by combining next-generation sequencing (RNA- and bisulfite sequencing), super-resolution microscopy, and immunofluorescence staining at 7days post injury. We observed significant transcriptomic changes primarily enriched in neuroinflammation and synaptogenesis associated pathways. Transcription factors (TFs) that regulate neurogenesis and neuron plasticity, including Egr1, Klf4, and Myc, are significantly upregulated. Along with global changes in chromatin arrangements and DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), bisulfite sequencing further reveals the involvement of DNA methylation changes in regulating cytokine, growth factor, and ion channel expression. Collectively, our results pave the way towards understanding transcriptomic and epigenomic mechanism in conferring long-term disease risks at distal tissues away from the primary lesion center and shed light on potential molecular targets that govern the regulatory mechanism at distal spinal cord tissues.

Illuminating the biosynthesis pathway genes involved in bioactive specific monoterpene glycosides in Paeonia veitchii Lynch by a combination of sequencing platforms

BackgroundPaeonia veitchii Lynch, a well-known herb from the Qinghai-Tibet Plateau south of the Himalayas, can synthesize specific monoterpene glycosides (PMGs) with multiple pharmacological activities, and its rhizome has become an indispensable ingredient in many clinical drugs. However, little is known about the molecular background of P. veitchii, especially the genes involved in the biosynthetic pathway of PMGs.ResultsA corrective full-length transcriptome with 30,827 unigenes was generated by combining next-generation sequencing (NGS) and single-molecule real-time sequencing (SMRT) of six tissues (leaf, stem, petal, ovary, phloem and xylem). The enzymes terpene synthase (TPS), cytochrome P450 (CYP), UDP-glycosyltransferase (UGT), and BAHD acyltransferase, which participate in the biosynthesis of PMGs, were systematically characterized, and their functions related to PMG biosynthesis were analysed. With further insight into TPSs, CYPs, UGTs and BAHDs involved in PMG biosynthesis, the weighted gene coexpression network analysis (WGCNA) method was used to identify the relationships between these genes and PMGs. Finally, 8 TPSs, 22 CYPs, 7 UGTs, and 2 BAHD genes were obtained, and these putative genes were very likely to be involved in the biosynthesis of PMGs. In addition, the expression patterns of the putative genes and the accumulation of PMGs in tissues suggested that all tissues are capable of biosynthesizing PMGs and that aerial plant parts could also be used to extract PMGs.ConclusionWe generated a large-scale transcriptome database across the major tissues in P. veitchii, providing valuable support for further research investigating P. veitchii and understanding the genetic information of plants from the Qinghai-Tibet Plateau. TPSs, CYPs, UGTs and BAHDs further contribute to a better understanding of the biology and complexity of PMGs in P. veitchii. Our study will help reveal the mechanisms underlying the biosynthesis pathway of these specific monoterpene glycosides and aid in the comprehensive utilization of this multifunctional plant.

Arabinosyltransferase C Mediates Multiple Drugs Intrinsic Resistance by Altering Cell Envelope Permeability in Mycobacterium abscessus.

ABSTRACTMycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus.IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

Genomic prediction of preliminary yield trials in chickpea: Effect of functional annotation of SNPs and environment.

Achieving yield potential in chickpea (Cicer arietinum L.) is limited by many constraints that include biotic and abiotic stresses. Combining next-generation sequencing technology with advanced statistical modeling has the potential to increase genetic gain efficiently. Whole genome resequencing data was obtained from 315 advanced chickpea breeding lines from the Australian chickpea breeding program resulting in more than 298,000 single nucleotide polymorphisms (SNPs) discovered. Analysis of population structure revealed a distinct group of breeding lines with many alleles that are absent from recently released Australian cultivars. Genome-wide association studies (GWAS) using these Australian breeding lines identified 20 SNPs significantly associated with grain yield in multiple field environments. A reduced level of nucleotide diversity and extended linkage disequilibrium suggested that some regions in these chickpea genomes may have been through selective breeding for yield or other traits. A large introgression segment that introduced from C. echinospermum for phytophthora root rot resistance was identified on chromosome 6, yet it also has unintended consequences of reducing yield due to linkage drag. We further investigated the effect of genotype by environment interaction on genomic prediction of yield. We found that the training set had better prediction accuracy when phenotyped under conditions relevant to the targeted environments. We also investigated the effect of SNP functional annotation on prediction accuracy using different subsets of SNPs based on their genomic locations: regulatory regions, exome, and alternative splice sites. Compared with the whole SNP dataset, a subset of SNPs did not significantly decrease prediction accuracy for grain yield despite consisting of a smaller number of SNPs.

Molecular Characteristics of Escherichia coli Causing Bloodstream Infections During 2010-2015 in a Tertiary Hospital, Shanghai, China.

BackgroundThe bloodstream infections (BSI) caused by Escherichia coli pose a serious threat to human health. To explore molecular characteristics of E. coli causing BSI, we collected E. coli isolates causing BSI in Huashan Hospital, Shanghai, China during 2010–2015.MethodsIn all E. coli isolates causing BSI collected from this study, polymerase chain reaction (PCR) was used to detect ESBLs and carbapenemase genes, and minimum inhibitory concentrations (MICs) were determined with agar dilution method. Outer membrane proteins were examined by SDS-PAGE in carbapenem-resistant strains. The genetic background of blaKPC gene was investigated by combining next-generation sequencing with a PCR mapping approach. Conjugation and transformation experiments were performed to verify the mobilization of blaKPC. The transcription levels of the blaKPC gene were measured by RT-PCR.ResultsDuring 2010–2015, a total of 207 E. coli BSI strains were isolated. The positive rates of β-lactamase resistant genes were 0.48% (blaKPC), 57% (blaTEM), 23.67% (blaCTX-M-1), 18.84% (blaCTX-M-9), and 1.93% (blaSHV). High rates of blaTEM, blaCTX-M-1, and blaCTX-M-9 were consistent with the poor activity of third-generation cephalosporins and aztreonam in vitro, except for carbapenem and β-lactamase inhibitor combinations. Low susceptibility rates were observed for piperacillin (25.1%) in contrast to the increased susceptibility when combined with β-lactamase inhibitors, namely piperacillin-tazobactam (90.8%). Only one KPC-producing E. coli strain was detected. Despite the combination of OmpC loss, the low expression level of KPC may be responsible for its lower resistance to carbapenems compared to E. coli DH5α (pKP12-100).ConclusionE. coli strains isolated from BSI were still highly susceptible to carbapenems and β-lactamase inhibitor combinations, and blaCTX-M was the dominant genotype of ESBLs. The low expression of blaKPC may be the reason for the low resistance to carbapenems.

Complete genome sequence of a divergent sweet potato chlorotic stunt virus isolate infecting Calystegia hederacea in China.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) is one of the most destructive viruses infecting sweet potatoes. In this study, we determined the complete genome sequence of an SPCSV-like isolate (CH) from Calystegia hederacea Wall. (Convolvulaceae), a weed species related to sweet potato, by combining next-generation sequencing and rapid amplification of cDNA ends. Comparisons of genome sequences and organization confirmed the classification of CH as SPCSV. However, the sequences and phylogenetic data revealed substantial genetic divergence between CH and all known SPCSV isolates. The amino acid sequence identity between the putative proteins in SPCSV-CH and the corresponding proteins in other known SPCSV isolates in each case was less than 85.0%. Phylogenetic analysis indicated that SPCSV-CH is separate from the groups of the known SPCSV isolates. Additionally, SPCSV-CH RNA1 lacks a p22 gene. A 10.1-kDa putative protein (p10) encoded by a sequence in the 5'-terminal region of RNA2 in SPCSV-CH is much larger than the corresponding protein in all known SPCSV isolates.

MET alterations in biphasic squamoid alveolar papillary renal cell carcinomas and clinicopathological features.

Biphasic squamoid alveolar papillary renal cell carcinoma (BSA-PRCC) is a recently studied lesion considered a morphologic variant of papillary renal cell carcinoma (RCC), more closely related to type 1. Considering the role of proto-oncogene MET in both sporadic type 1 papillary RCC and hereditary papillary RCC, we aimed to explore the role of MET activation in the oncogenesis of BSA-PRCC. We identified 17 patients with either unique (n = 14) or multiple (n = 3) BSA-PRCC, all localized, and performed an integrative analysis of MET status in 18 formalin-fixed paraffin-embedded tumors combining next-generation sequencing analysis, fluorescent in situ hybridization and immunohistochemistry. Trisomy 7 was found in 86% of tumors (14/16) without MET amplification at 7q31 (15/15). A pathogenic MET genetic variant was identified in 60% (9/15) of cases, at the germline level in 57% (4/7) of tested patients or at the somatic level (5/11). MET expression was observed in all tumors with a higher value of combined score in large cells (mean 97%, range 80-100%) than in small cells (mean 74%, range 10-100%) and was lower in two cases without MET copy number gain. In conclusion, our study provides additional evidence to consider biphasic squamoid alveolar papillary RCC as a morphological variant of type 1 papillary renal RCC. Our data strongly suggest that MET represents a major oncogenic driver gene in BSA-PRCC, harboring a higher frequency of MET mutation that encourages to further explore the benefice of anti-MET targeted therapies for aggressive BSA-PRCC.

Genetic architecture of leaf morphological and physiological traits in a Populus deltoides ‘Danhong’ × P. simonii ‘Tongliao1’ pedigree revealed by quantitative trait locus analysis

Understanding the genetic architecture of leaf morphological and physiological traits will help plant breeders develop high biomass poplar genotypes. Quantitative trait locus (QTL) studies combining next-generation sequencing techniques can advance our understanding of the genetic basis of complex traits. In this study, we measured 13 leaf morphological and physiological traits and identified quantitative trait loci (QTLs) in a Populus deltoides ‘Danhong’ × P. simonii ‘Tongliao1’ F1 population (500 progenies) using a high-density genetic map constructed by whole genome re-sequencing. This linkage map consisted of 5796 single nucleotide polymorphism (SNP) markers assigned to 19 linkage groups (LGs), spanning 2683.80 centimorgans (cM) of genetic length, with an average marker density of 0.46 cM. We identified 109 QTLs on 18 LGs for leaf morphological traits and 55 QTLs on 14 LGs for leaf physiological traits. One-hundred eight putative candidate genes were identified within the candidate genomic region. Co-expression network and gene ontology enrichment analyses suggested that these candidate genes were involved in the photosynthetic process. The differential expression patterns of the CYCLIN (Potri.015G112200) and RED CHLOROPHYLL REDUCTASE (Potri.007G043600) genes between two parents indicated their potential roles in leaf morphological and physiological traits. These findings decipher the genetic architecture of leaf morphological and physiological traits in the P. deltoides ‘Danhong’ × P. simonii ‘Tongliao1’ pedigree and provide candidate genes for future poplar genetic improvement.

Sequential mutational evaluation of CALR -mutated myeloproliferative neoplasms with thrombocytosis reveals an association between CALR allele burden evolution and disease progression.

In myeloproliferative neoplasms (MPN), JAK2V617F allele burden measurement has an impact on prognosis that helps in patient monitoring. Less is known about its usefulness in CALR-mutated cases. Additional mutations found by next-generation sequencing have also shown an impact on prognosis that may drive therapeutic choices, especially in myelofibrosis, but few studies focused on CALR-mutated patients. We performed a molecular evaluation combining next-generation sequencing with a myeloid panel and CALR allele burden measurement at diagnosis and during follow-up in a cohort of 45 patients with CALR-mutated essential thrombocythaemia. The bone marrow histology was also blindly reviewed in order to apply the WHO2016 classification. The most frequently mutated gene was TET2 (11/21 mutations). CALR type 1-like patients appear to have a more complex molecular landscape. We found an association between disease progression and CALR allele burden increase during follow-up, independently of additional mutations and WHO2016-reviewed diagnosis. Patients with disease progression at the time of follow-up showed a significant increase in CALR allele burden (+16·7%, P=0·005) whereas patients without disease progression had a stable allele burden (+3·7%, P=0·194). This result argues for clinical interest in CALR allele burden monitoring.

Combining next-generation sequencing and single-molecule sequencing to explore brown plant hopper responses to contrasting genotypes of japonica rice

BackgroundThe brown plant hopper (BPH), Nilaparvata lugens, is one of the major pest of rice (Oryza sativa). Plant defenses against insect herbivores have been extensively studied, but our understanding of insect responses to host plants’ resistance mechanisms is still limited. The purpose of this study is to characterize transcripts of BPH and reveal the responses of BPH insects to resistant rice at transcription level by using the advanced molecular techniques, the next-generation sequencing (NGS) and the single-molecule, real-time (SMRT) sequencing.ResultsThe current study obtained 24,891 collapsed isoforms of full-length transcripts, and 20,662 were mapped to known annotated genes, including 17,175 novel transcripts. The current study also identified 915 fusion genes, 1794 novel genes, 2435 long non-coding RNAs (lncRNAs), and 20,356 alternative splicing events. Moreover, analysis of differentially expressed genes (DEGs) revealed that genes involved in metabolic and cell proliferation processes were significantly enriched in up-regulated and down-regulated sets, respectively, in BPH fed on resistant rice relative to BPH fed on susceptible wild type rice. Furthermore, the FoxO signaling pathway was involved and genes related to BPH starvation response (Nlbmm), apoptosis and autophagy (caspase 8, ATG13, BNIP3 and IAP), active oxygen elimination (catalase, MSR, ferritin) and detoxification (GST, CarE) were up-regulated in BPH responses to resistant rice.ConclusionsThe current study provides the first demonstrations of the full diversity and complexity of the BPH transcriptome, and indicates that BPH responses to rice resistance, might be related to starvation stress responses, nutrient transformation, oxidative decomposition, and detoxification. The current result findings will facilitate further exploration of molecular mechanisms of interaction between BPH insects and host rice.

Targeted Gene Next-Generation Sequencing Panel in Patients with Advanced Lung Adenocarcinoma: Paving the Way for Clinical Implementation.

Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (κ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.

PO-496 Loss of PARG drives PARP inhibitor resistance in BRCA2-deficient mouse mammary tumours

IntroductionInhibitors of poly(ADP-ribose) polymerase (PARP) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, resistance to PARP inhibitors (PARPi) is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway.Material and methodsTo tackle this question, we generated matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumours. By combining next-generation sequencing with functional genetic screens, we identified loss of poly(ADP-ribose) glycohydrolase (PARG) as a major resistance mechanism.Results and discussionsWe demonstrate that PARG depletion restores PAR formation, rescues controlled DNA replication fork progression and promotes the recruitment of downstream DNA repair factors. The potential relevance of PARG in clinical PARPi resistance is underscored by the presence of PARG-negative clones in a subset of human triple-negative breast and serous ovarian cancers. Importantly, acquisition However, the gain of PARPi resistance comes at a cost, as PARG inactivation results in new vulnerabilities that can be exploited therapeutically.ConclusionWe conclude that loss of PARG should be assessed as a potential cause of clinical PARPi resistance. In this case, measurement of PARG activity should further improve clinical decision making for patients with tumours that lack homology-directed DNA repair.

Combining targeted panel-based resequencing and copy-number variation analysis for the diagnosis of inherited syndromic retinopathies and associated ciliopathies

Inherited syndromic retinopathies are a highly heterogeneous group of diseases that involve retinal anomalies and systemic manifestations. They include retinal ciliopathies, other well-defined clinical syndromes presenting with retinal alterations and cases of non-specific multisystemic diseases. The heterogeneity of these conditions makes molecular and clinical characterization of patients challenging in daily clinical practice. We explored the capacity of targeted resequencing and copy-number variation analysis to improve diagnosis of a heterogeneous cohort of 47 patients mainly comprising atypical cases that did not clearly fit a specific clinical diagnosis. Thirty-three likely pathogenic variants were identified in 18 genes (ABCC6, ALMS1, BBS1, BBS2, BBS12, CEP41, CEP290, IFT172, IFT27, MKKS, MYO7A, OTX2, PDZD7, PEX1, RPGRIP1, USH2A, VPS13B, and WDPCP). Molecular findings and additional clinical reassessments made it possible to accurately characterize 14 probands (30% of the total). Notably, clinical refinement of complex phenotypes was achieved in 4 cases, including 2 de novo OTX2-related syndromes, a novel phenotypic association for the ciliary CEP41 gene, and the co-existence of biallelic USH2A variants and a Koolen-de-Vries syndrome–related 17q21.31 microdeletion. We demonstrate that combining next-generation sequencing and CNV analysis is a comprehensive and useful approach to unravel the extensive phenotypic and genotypic complexity of inherited syndromic retinopathies.

Dietary Analysis of Marine Fish Species: Enhancing the Detection of Prey-Specific DNA Sequences via High-Throughput Sequencing Using Blocking Primers

Dietary studies of fish can enhance our understanding of predator-prey interactions and provide important insights into complex marine food webs. In this study, we performed dietary analyses of three commercially important fish species in South China: Acanthopagrus latus, Pampus argenteus, and Scomberomorus commerson. Two distinct fragments of the mitochondrial cytochrome c oxidase I (mtCOI) gene, a small (130 bp) and large (300 bp) amplicon, were amplified from the DNA extracted from the gut contents in the presence of blocking primers designed to inhibit the amplification of predator-specific DNA. The amplicons from each predator species were tagged with a unique indexing bar code and sequenced using the Illumina MiSeq platform. Our results showed that the three fish species consumed a wide variety of prey, including jellyfish, copepods, crustaceans, and fish. Seasonal variations in prey composition and species-selective predation were observed. Our approach of combining next-generation sequencing with blocking primers greatly enhanced the speed and resolution of the dietary analyses in marine fishes and could provide novel insights into marine ecosystems.

Climatic niche evolution is faster in sympatric than allopatric lineages of the butterfly genus Pyrgus.

Understanding how speciation relates to ecological divergence has long fascinated biologists. It is assumed that ecological divergence is essential to sympatric speciation, as a mechanism to avoid competition and eventually lead to reproductive isolation, while divergence in allopatry is not necessarily associated with niche differentiation. The impact of the spatial context of divergence on the evolutionary rates of abiotic dimensions of the ecological niche has rarely been explored for an entire clade. Here, we compare the magnitude of climatic niche shifts between sympatric versus allopatric divergence of lineages in butterflies. By combining next-generation sequencing, parametric biogeography and ecological niche analyses applied to a genus-wide phylogeny of Palaearctic Pyrgus butterflies, we compare evolutionary rates along eight climatic dimensions across sister lineages that diverged in large-scale sympatry versus allopatry. In order to examine the possible effects of the spatial scale at which sympatry is defined, we considered three sets of biogeographic assignments, ranging from narrow to broad definition. Our findings suggest higher rates of niche evolution along all climatic dimensions for sister lineages that diverge in sympatry, when using a narrow delineation of biogeographic areas. This result contrasts with significantly lower rates of climatic niche evolution found in cases of allopatric speciation, despite the biogeographic regions defined here being characterized by significantly different climates. Higher rates in allopatry are retrieved when biogeographic areas are too widely defined-in such a case allopatric events may be recorded as sympatric. Our results reveal the macro-evolutionary significance of abiotic niche differentiation involved in speciation processes within biogeographic regions, and illustrate the importance of the spatial scale chosen to define areas when applying parametric biogeographic analyses.