Target Sequencing Research Articles

Abstract 12364: Dysfunction of N-terminal Acetylation Causes Multiple Electrical Abnormalities Leading to Long QT Syndrome and Dilated Cardiomyopathy

Introduction: NAA10-related syndrome, also known as Ogden syndrome, is characterized by long QT syndrome (LQTS), cardiomyopathy, hypotonia, and neurodevelopmental delay. NAA10 complexes with the regulatory subunit NAA15 to make the post-translationally modifying N-terminal acetyltransferase, NatA. Despite the association of LQTS and NAA10 mutations, the mechanism is unknown. Hypothesis: NAA10 dysfunction affects the function of cardiac ion channels or related proteins causing LQTS. Methods: Target sequencing of large kindred with multiple family members who had LQTS and sudden death revealed a segregating mutation in NAA10 (c.10C>A; p.R4S). Somatic cell reprogramming was used to create an induced pluripotent stem cell (iPSC) line from a gene-positive male patient with LQTS and dilated cardiomyopathy, (pNAA10 R4S ). We also genome-edited the mutation into a control iPSC line (eNAA10 R4S ). The iPSC lines were differentiated into cardiomyocytes (iPSC-CMs) for electrophysiologic characterization. Results: Action potential duration (APD) of wild-type (WT) and mutant iPSC-CMs was evaluated using patch-clamp and dynamic-clamp electrophysiology. There was significant APD prolongation in NAA10 R4S -iPSC-CMs and eNAA10 R4S -iPSC-CMs as compared to WT iPSC-CMs (448.1 ± 102.2 ms and 392.3 ± 20.8 ms, vs 73.1 ± 12.1 ms; p<0.05). We then investigated potential derangements in the underlying sodium (I Na ), potassium (I K ) and calcium (I CaL ) currents. Voltage-clamp measurements demonstrated increased peak I Na in mutant iPSC-CMs lines (pNAA10 R4S : -72.2 ± 11.1 pA/pF and eNAA10 R4S : -77.4 ± 15.3 pA/pF vs. WT: -44.4 ± 7.9 pA/pF, N>12, p<0.05) However, there were no changes in voltage-dependent activation or inactivation. Similarly peak I CaL was also increased (-10.2 ± 1.7 pA/pF, vs -7.0 ± 1.2 pA/pF (N= 5 and 3, respectively)) compared to control iPSC-CMs without any changes in Ca 2+ channel biophysics. There were no significant differences in measured I K . Conclusion: We demonstrate that the NAA10 mutation cause APD prolongation by increasing I Na and I CaL current densities leading to QT prolongation and arrhythmia. This data indicated a novel role for N-terminal acetylation in cardiac ion channel regulation leading to cardiovascular disorders.

Upregulation of CD47 Expression in De Novo Diffuse Large B-Cell Lymphoma Is More Frequent in Activated B-Cell Type

CD47 is a marker of self that provides a “don't-eat-me-signal” through activation of signal-regulatory protein alpha (SIRPa), a cell surface receptor expressed on monocytes/macrophages and granulocytes. This interaction negatively regulates effector functions such as, phagocytosis, migration, and superoxide production. Upregulation of CD47 expression in cancer, including diffuse large B-cell lymphoma (DLBCL), has emerged as a mechanism to escape innate immune surveillance. Using conventional immunohistochemical detection, we assessed CD47 expression in DLBCL and interrogated its association with clinicopathologic features.Patients with de novo DLBCL were identified from two large institutions and were uniformly treated with R-CHOP and had sufficient material for study. Immunohistochemical stains (IHC) were performed on FFPE tissue (Hans algorithm, BCL2, MYC, and CD47) and scored semi-quantitatively from no reactivity (0) to strong (2 and 3; Figure 1). Mutational analysis using a 334 gene target sequencing panel, gene expression profiling using Lymph2Cx to determine the cell of origin (COO), and FISH analysis for MYC, BCL2, and BCL6 translocations, were performed. The Lymphgen tool (Wright et al, 2020) was also used to determine the DLBCL group. Fisher's exact test and Kaplan-Meier survival analysis for overall survival (OS) were performed and P <0.05 was considered significant.CD47 expression was assessed by IHC in a cohort of 152 cases of de novo DLBCL, including 107 cases of germinal center B-cell (GCB) type (70%), 37 cases of activated B-cell (ABC) type (24%), and 8 cases of intermediate type (5%). A total of 17 cases (11%) showed strong and diffuse CD47 expression with IHC scores of 2 or above (CD47hi). CD47hi cases were significantly more frequent in ABC DLBCL (24%, 9/37) than GCB DLBCL (6%, 6/107; P=0.003). The remaining 2 CD47hi cases were in the intermediate DLBCL group (25%, 2/8). ABC DLBCL with CD47hi showed more frequent mutations with TET2 (33% vs 7%; P=0.08) and ZFP36L1 (22% vs 0%; P=0.05) compared to cases with low expression of CD47 with IHC scores of less than 2 (CD47low). ABC DLBCL with CD47low showed more frequent mutations of NOTCH2 (18% vs 0%; P=0.31) and MYD88 (29% vs 11%; P=0.4) compared to CD47hi. GCB DLBCL with CD47hi showed frequent mutations of TP53 (67% vs 21%; P=0.026) and CCND1 (33% vs 0%; P=0.003) compared to CD47low. None of the 13 cases with double- or triple-hit for MYC, BCL2 and/or BCL6 showed CD47 expression. The Lymphgen tool showed that cases of DLBCL with CD47hi were mostly in the ‘other’ group (50%), with other groups represented such as ST2 (21%), EZB (14%), MCD and BN2 (1 case each). There was no difference in overall survival (OS) between CD47hi and CD47low DLBCL (5-year OS, 75% vs 72%; P=0.57), or with the GCB or ABC subtypes.Strong expression with CD47 is more frequent in ABC DLBCL and is seen in a subset of GCB DLBCL with mutations in TP53 and/or CCND1. The level of CD47 expression does not appear to predict OS in patients with DLBCL treated with R-CHOP. This study demonstrates that conventional immunohistochemical methods can readily identify DLBCL with high CD47 expression, and these patients may benefit from the use of anti-CD47 therapy. [Display omitted] DisclosuresHerrera: Gilead Sciences: Research Funding; Takeda: Consultancy; Tubulis: Consultancy; Karyopharm: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Seagen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding.

IsoCNV: in silico optimization of copy number variant detection from targeted or exome sequencing data

BackgroundAccurate copy number variant (CNV) detection is especially challenging for both targeted sequencing (TS) and whole‐exome sequencing (WES) data. To maximize the performance, the parameters of the CNV calling algorithms should be optimized for each specific dataset. This requires obtaining validated CNV information using either multiplex ligation-dependent probe amplification (MLPA) or array comparative genomic hybridization (aCGH). They are gold standard but time-consuming and costly approaches.ResultsWe present isoCNV which optimizes the parameters of DECoN algorithm using only NGS data. The parameter optimization process is performed using an in silico CNV validated dataset obtained from the overlapping calls of three algorithms: CNVkit, panelcn.MOPS and DECoN. We evaluated the performance of our tool and showed that increases the sensitivity in both TS and WES real datasets.ConclusionsisoCNV provides an easy-to-use pipeline to optimize DECoN that allows the detection of analysis-ready CNV from a set of DNA alignments obtained under the same conditions. It increases the sensitivity of DECoN without the need for orthogonal methods. isoCNV is available at https://gitlab.com/sequentiateampublic/isocnv.

Development of a Panel of Genotyping-in-Thousands by Sequencing in Capsicum.

Genotyping by sequencing (GBS) enables genotyping of multiple loci at low cost. However, the single nucleotide polymorphisms (SNPs) revealed by GBS tend to be randomly distributed between individuals, limiting their direct comparisons without applying the various filter options to obtain a comparable dataset of SNPs. Here, we developed a panel of a multiplex targeted sequencing method, genotyping-in-thousands by sequencing (GT-seq), to genotype SNPs in Capsicum spp. Previously developed Fluidigm® SNP markers were converted to GT-seq markers and combined with new GT-seq markers developed using SNP information obtained through GBS. We then optimized multiplex PCR conditions: we obtained the highest genotyping rate when the first PCR consisted of 25 cycles. In addition, we determined that 101 primer pairs performed best when amplifying target sequences of 79 bp. We minimized interference of multiplex PCR by primer dimer formation using the PrimerPooler program. Using our GT-seq pipeline on Illumina Miseq and Nextseq platforms, we genotyped up to 1,500 (Miseq) and 1,300 (Nextseq) samples for the optimum panel size of 100 loci. To allow the genotyping of Capsicum species, we designed 332 informative GT-seq markers from Fluidigm SNP markers and GBS-derived SNPs. This study illustrates the first application of GT-seq in crop plants. The GT-seq marker set developed here will be a useful tool for molecular breeding of peppers in the future.

Clinical Outcomes of Genotype-Matched Therapy for Recurrent Gynecological Cancers: A Single Institutional Experience.

Recent advances in next-generation sequencing and genome medicine have contributed to treatment decisions in patients with cancer. Most advanced gynecological cancers develop resistance to chemotherapy and have a poor prognosis. Therefore, we conducted genomic tests in gynecological tumors to examine the efficacy and clinical feasibility of genotype-matched therapy. Target sequencing was performed in 20 cases of gynecological cancers (cervical cancer, 6; endometrial cancer, 6; and ovarian cancer, 6). Both actionable and druggable genes were identified in 95% (19/20) of the cases. Among them, seven patients (35%) received genotype-matched therapy, which was effective in three patients. Of the three patients, one patient with a PTEN mutation received everolimus, another patient with a TSC2 mutation received everolimus and letrozole, and the patient with a BRIP1 mutation received olaparib. Subsequently, disease control in these three patients lasted for more than half a year. However, all patients relapsed between 9 and 13 months after the initiation of genotype-matched therapy. In this study, the response rate of genotype-matched therapy was 43% (3/7), which may have contributed to improved prognoses. Therefore, genotype-matched therapies may help patients with refractory gynecological cancers achieve better outcomes.

Emery-Dreifuss muscular dystrophy presenting as a heart rhythm disorders in children

Abstract Introduction Emery-Dreifuss muscular dystrophy (EDMD) is an inherited muscle dystrophy often accompanied by cardiac abnormalities in the form of supraventricular arrhythmias, conduction defects, sinus node dysfunction. Cardiac phenotype typically arises years after skeletal muscle presentations, though, can be severe and life-threatening. The disease usually manifests during the third decade of life with elbow joint contractions and progressive muscle weakness and atrophy. Objective To present our clinical experience of diagnosis and treatment of arrhythmias in children with Emery-Dreifuss muscular dystrophy Materials and methods We enrolled 5 patients with different forms of EDMD (X-linked and autosomal dominant) linked to the mutations in EMD and LMNA genes, presented with early onset of cardiac abnormalities and no leading skeletal muscle phenotype. The predominant forms of cardiac pathology were atrial flutter, atrial fibrillation and conduction disturbances that progress over time. Clinical examination included physical examination, 12-lead electrocardiography, Holter ECG monitoring (HM), transthoracic echocardiography, neurological examination and biochemical and hormone tests. Also we performed CMR, electrophysiological study (EPS), treadmill test of some patients. One patient underwent an endomyocardial biopsy to exclude inflammatory heart disease. Target sequencing was performed using a panel of 108 or 172 genes Results We observed five patients with EDMD and cardiac debut during first-second decades of life: 3 with 1st subtype (variants in EMD gene) and 2 with 2nd subtype (variants in LMNA gene). All patients were males. The mean age of cardiac manifestation was 13,2±3,11 (from 9 to 16 y.o.). The mean follow-up period was 7,4±2,6 years. All patients presented with sinus node dysfunction and four out of five with AV conduction abnormalities. The leading arrhythmic phenotypes included various types of supraventricular arrhythmias: multifocal atrial tachycardia (AT) (n=4), premature atrial captures (PACs) (n=4), atrial flutter, (AF) (n=3), atrial fibrillation (AFib) (n=3) and AV nodal recurrent tachycardia (AVRNT). Heart rhythm disorders were the first manifestation in all three patients with 1st EDMD subtype. Radiofrequency ablation was performed in 2 patients, one of them received permanent pacemaker implantation. Conclusions In conclusion, while being the rare cases, heart rhythm disorders can represent the first and for a long time, the only clinical symptom of EDMD even in the pediatric group of patients. Therefore, thorough laboratory and neurological screening along with genetic studies, are of importance in each pediatric patient presenting with complex heart rhythm disorders of primary supraventricular origin to exclude EDMD or other neuromuscular disorders. Funding Acknowledgement Type of funding sources: None.

Identification and validation of tissue or ctDNA PTPRD phosphatase domain deleterious mutations as prognostic and predictive biomarkers for immune checkpoint inhibitors in non-squamous NSCLC

BackgroundWith the revolutionary progress of immune checkpoint inhibitors (ICIs) achieved in non-small cell lung cancers (NSCLC), identifying patients benefiting from ICIs becomes critical and urgent. The associations of genomic alterations in protein tyrosine phosphatase receptor-type (PTPRs) and ICIs responses are unknown.MethodsWhole-exome sequencing (WES) of 73 advanced NSCLC tumors sampled before anti-PD-(L)1 therapy was carried out with corresponding clinical data collected as a discovery cohort to find the associations of PTPR mutations and ICI responses. Three validation cohorts consolidated by 7 public cohorts of 1920 NSCLC patients with WES or target sequencing data of tumor tissue-derived DNA or circulating tumor DNA (ctDNA) and relevant clinical data were applied as validation cohorts. The lung adenocarcinoma (LUAD) cohort (n=586) in The Cancer Genome Atlas (TCGA) database was used for analyzing the potential anti-tumor immunologic mechanisms.ResultsWith the highest mutation frequency among all PTPRs, PTPRD mutations in non-squamous NSCLC (ns-NSCLC) were linked to longer progression-free survivals (PFS, 324 vs 63 days, hazard ratio (HR)=0.36, p= 0.0152) and higher objective response rate (ORR, p=0.0099). In validation cohort 1 (n=377), ns-NSCLC patients with tissue PTPRD mutations had favorable PFS (9.10 vs 4.33 months, HR=0.62, p=0.0184) and ORR (p=0.013). In validation cohort 2 (n=406), ns-NSCLC patients with tissue PTPRD mutations had favorable overall survivals (OS, over 40 vs 11.94 months, HR=0.57, p=0.011). In validation cohort 3 (n=1137), ns-NSCLC patients with ctDNA PTPRD mutations had longer PFS (6.97 vs 2.73 months, HR=0.63, p=0.028) and higher ORR (p=0.047). Moreover, it was deleterious mutations in phosphatase domains (phosphatase-mut), rather than other mutations (other-mut), that were responsible of PTPRD’s prediction efficiency. In addition, in validation cohort 3, ctDNA phosphatase-mut also functioned as a predictive biomarker helping identify patients benefiting more from ICIs than chemotherapy (interaction P for PFS=0.0506, for OS=0.04). Univariate and multivariate regression analysis revealed that phosphatase-mut was independent on PD-L1 expression and tumor mutation burden (TMB) to predict. In silico analysis based on TCGA LUAD cohort discovered enhanced anti-tumor immunity in phosphatase-mut patients.ConclusionsTissue or ctDNA PTPRD phosphatase domain deleterious mutations might function as a both prognostic and predictive biomarker predicting clinical outcomes of ICIs in ns-NSCLC patients, independent on TMB or PD-L1 expression.

Upregulation of GBP1 in thyroid primordium is required for developmental thyroid morphogenesis

PurposeCongenital hypothyroidism (CH) is a common congenital endocrine disorder in humans. CH-related diseases such as athyreosis, thyroid ectopy, and hypoplasia are primarily caused by dysgenic thyroid development. However, the underlying molecular mechanisms remain unknown. MethodsTo identify novel CH candidate genes, 192 CH patients were enrolled, and target sequencing of 21 known CH-related genes was performed. The remaining 98 CH patients carrying no known genes were subjected to exome sequencing (ES). The functions of the identified variants were confirmed using thyroid epithelial cells in vitro and in zebrafish model organisms in vivo. ResultsFour pathogenic GBP1 variations from three patients were identified. In zebrafish embryos, gbp1 knockdown caused defective thyroid primordium morphogenesis and hypothyroidism. The thyroid cells were stuck together and failed to dissociate from each other to form individual follicles in gbp1-deficient embryos. Furthermore, defects were restored with wild-type human GBP1 (hGBP1) messenger RNA (mRNA) except for mutated hGBP1 (p.H150Y, p.L187P) overexpression. GBP1 promoted β-catenin translocation into the cytosol and suppressed the formation of cellular adhesion complexes. Suppression of cell–cell adhesion restored the thyroid primordium growth defect observed in gbp1-deficient zebrafish embryos. ConclusionThis study provides further understanding regarding thyroid development and shows that defective cellular remodeling could cause congenital hypothyroidism.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

High genetic diversity and superinfection by Anaplasma marginale strains in naturally infected Angus beef cattle during a clinical anaplasmosis outbreak in southeastern Brazil

Anaplasma marginale is an obligate intracellular Gram-negative bacterium that is parasitic to erythrocytes and is the main agent of bovine anaplasmosis. This disease causes severe anemia and reduces weight gain and milk production, thus giving rise to major economic losses relating to livestock worldwide. The genetic diversity of this bacterium has been characterized based on sequences of major surface proteins (MSPs), especially MSP1α. This has enabled identification of several geographical strains, according to different amino acid sequences. The aim of this study was to investigate the genetic diversity of A. marginale in naturally infected Angus beef cattle during a disease outbreak in southeastern Brazil. Four blood samples were collected over a four-month period from each of 20 animals on a cattle farm in Itú, São Paulo, Brazil. Serum samples were subjected to indirect ELISA to detect anti-A. marginale IgG antibodies. The 80 whole-blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1β gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing using the Sanger method. The sequences obtained were analyzed for genetic diversity using the RepeatAnalyzer software. Both iELISA tests, using recombinant MSP5 and the Anaplasma antibody test kit (VMRD), revealed high seroprevalence: 91.25% and 97.5%, respectively. In qPCR, 100% of the samples were positive, with between 103 and 107 DNA copies/μL. In the snPCR based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis on the 36 sequences obtained showed the presence of genotypes H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). The RepeatAnalyzer software identified 36 strains in the study region, among which some had not previously been described in the literature (13 27 13 27 13 F; 16 FF; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91). High genetic diversity of A. marginale bacteria was found on this farm in Itú, São Paulo.

Nicotinamide nucleotide transhydrogenase mutation analysis in Chinese patients with thyroid dysgenesis.

Thyroid dysgenesis (TD) accounts for 80% cases of congenital hypothyroidism, which is the most common neonatal disorder. Until now, the gene mutations have been reported associated with TD can only account for 5% cases, suggesting the genetic heterogeneity of the pathology. Nicotinamide nucleotide transhydrogenase (NNT) plays a crucial role in regulating redox homeostasis, patients carrying NNT mutations have been described with a clinical phenotype of hypothyroidism. As TD risk is increased in the context of several syndromes and redox homeostasis is vital for thyroid development and function, NNT might be a candidate gene involved in syndromic TD. Therefore, we performed target sequencing (TS) in 289 TD patients for causative mutations in NNT and conducted functional analysis of the gene mutations. TS and Sanger sequence were used to screen the novel mutations. For functional analysis, we performed western blot, measurement of NADPH/NADPtotal and H2 O2 generation, cell proliferation, and wounding healing assay. As a result, three presumably pathogenic mutations (c.811G > A, p.Ala271Ser; c.2078G > A, p.Arg693His; and c.2581G > A, p.Val861Met) in NNT had been identified. Our results showed the damaging effect of NNT mutations on stability and catalytic activity of proteins and redox balance of cells. In conclusion, our findings provided novel insights into the role of the NNT isotype in thyroid physiopathology and broaden the spectrum of pathogenic genes associated with TD. However, the pathogenic mechanism of NNT in TD is still need to be investigated in further study.

Rare and widespread: integrating Bayesian MCMC approaches, Sanger sequencing and Hyb-Seq phylogenomics to reconstruct the origin of the enigmatic Rand Flora genus Camptoloma.

Genera that are widespread, with geographically discontinuous distributions and represented by few species, are intriguing. Is their achieved disjunct distribution recent or ancient in origin? Why are they species-poor? The Rand Flora is a continental-scale pattern in which closely related species appear codistributed in isolated regions over the continental margins of Africa. Genus Camptoloma (Scrophulariaceae) is the most notable example, comprising three species isolated from each other on the northwest, eastern, and southwest Africa. We employed Sanger sequencing of nuclear and plastid markers, together with genomic target sequencing of 2190 low-copy nuclear genes, to infer interspecies relationships and the position of Camptoloma within Scrophulariaceae by using supermatrix and multispecies-coalescent approaches. Lineage divergence times and ancestral ranges were inferred with Bayesian Markov chain Monte Carlo (MCMC) approaches. The population history was estimated with phylogeographic coalescent methods. Camptoloma rotundifolium, restricted to Southern Africa, was shown to be a sister species to the disjunct clade formed by C. canariense, endemic to the Canary Islands, and C. lyperiiflorum, distributed in the Horn of Africa-Southern Arabia. Camptoloma was inferred to be sister to the mostly South African tribes Teedieae and Buddlejeae. Stem divergence was dated in the Late Miocene, while the origin of the extant disjunction was inferred as Early Pliocene. The current disjunct distribution of Camptoloma across Africa was likely the result of fragmentation and extinction and/or population bottlenecking events associated with historical aridification cycles during the Neogene; the pattern of species divergence, from south to north, is consistent with the "climatic refugia" Rand Flora hypothesis.

Genetic and functional analysis reveals TENM4 contributes to schizophrenia

SummaryTENM4, encoding a member of the teneurin protein family, is a risk gene shared by many types of mental diseases and is implicated in neuronal plasticity and signaling. However, the role and the mechanisms of TENM4 in schizophrenia (SCZ) remain unclear. We identified possible pathogenic mutations in the TENM4 gene through target sequencing of TENM4 in 68 SCZ families. We further demonstrated that aberrant expression of Ten-m leads to lower learning ability, sleep reduction, and increased aggressiveness in animal models. RNA sequencing showed that aberrant expression of Ten-m was related to stimulus perception and metabolic process, and Gene Ontology enrichment terms were neurogenesis and ATPase activity. This study provides strong evidence that TENM4 contributes to SCZ, and its functional mutations might be responsible for the impaired neural circuits and behaviors observed in SCZ.

Genome-Wide Comparative Analysis of Genetic Diversity of Regular and Specialty Maize Inbred Lines Through Genotyping by Target Sequencing (GBTS)

Genome-Wide Comparative Analysis of Genetic Diversity of Regular and Specialty Maize Inbred Lines Through Genotyping by Target Sequencing (GBTS)

Development of high-resolution multiple-SNP arrays for genetic analyses and molecular breeding through genotyping by target sequencing and liquid chip

Genotyping platforms, as critical supports for genomics, genetics, and molecular breeding, have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput, automatic, large-scale, and shared facilities. In this study, we integrated an improved genotyping by target sequencing (GBTS) system with capture-in-solution (liquid chip) technology to develop a multiple single-nucleotide polymorphism (mSNP) approach in which mSNPs can be captured from a single amplicon. From one 40K maize mSNP panel, we developed three types of markers (40K mSNPs, 251K SNPs, and 690K haplotypes), and generated multiple panels with various marker densities (1K–40K mSNPs) by sequencing at different depths. Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs. Compared with the one-amplicon-one-SNP system, mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection, linkage disequilibrium decay analysis, and genome-wide association studies. The technologies, protocols, and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals, plants, and microorganisms.

Paired analysis of tumor mutation burden calculated by targeted deep sequencing panel and whole exome sequencing in non-small cell lung cancer

Owing to rapid advancements in NGS (next generation sequen-cing), genomic alteration is now considered an essential pre-dictive biomarkers that impact the treatment decision in many cases of cancer. Among the various predictive biomarkers, tumor mutation burden (TMB) was identified by NGS and was con-sidered to be useful in predicting a clinical response in cancer cases treated by immunotherapy. In this study, we directly com-pared the lab-developed-test (LDT) results by target sequencing panel, K-MASTER panel v3.0 and whole-exome sequencing (WES) to evaluate the concordance of TMB. As an initial step, the reference materials (n = 3) with known TMB status were used as an exploratory test. To validate and evaluate TMB, we used one hundred samples that were acquired from surgically resected tissues of non-small cell lung cancer (NSCLC) patients. The TMB of each sample was tested by using both LDT and WES methods, which extracted the DNA from samples at the same time. In addition, we evaluated the impact of capture re-gion, which might lead to different values of TMB; the evalu-ation of capture region was based on the size of NGS and target sequencing panels. In this pilot study, TMB was evalu-ated by LDT and WES by using duplicated reference samples; the results of TMB showed high concordance rate (R2 = 0.887). This was also reflected in clinical samples (n = 100), which showed R2 of 0.71. The difference between the coding sequence ratio (3.49%) and the ratio of mutations (4.8%) indicated that the LDT panel identified a relatively higher number of mutations. It was feasible to calculate TMB with LDT panel, which can be useful in clinical practice. Furthermore, a customized approach must be developed for calculating TMB, which differs according to cancer types and specific clinical settings.

Virulence determinants and antimicrobial resistance of E. coli isolated from bovine clinical mastitis in some selected dairy farms of Bangladesh.

E. coli is one of the major significant pathogens causing mastitis, the most complex and costly diseases in the dairy industry worldwide. Present study was undertaken to isolate, detect the virulence factors, phylogroup, antimicrobial susceptibility and antimicrobial resistance genes in E. coli from cows with clinical mastitis. A total of 68 milk samples comprising 53 from clinical mastitis and 15 from apparently healthy cattle were collected from four different established dairy farms in Bangladesh. E. coli was isolated from the milk samples and identified by PCR targeting malB gene and sequencing of 16S rRNA gene. E. coli isolates were screened by PCR for the detection of major virulence genes (stx, eae and cdt) of diarrheagenic E. coli followed by phylogenetic grouping. Antimicrobial susceptibility of the E. coli isolates was determined by disk diffusion test and E. coli showing resistance was further screened for the presence of antimicrobial resistance genes. E. coli was isolated from 35.8% of the mastitis milk samples but none from the apparently healthy cattle milk. All the E. coli isolates were negative for stx, eae and cdt genes and belonged to the phylogenetic groups A and B1 which comprising of commensal E. coli. Antibiotic sensitivity testing revealed 84.2% (16/19) of the isolates as multidrug resistant. Highest resistance was observed against amoxicillin (94.5%) followed by ampicillin (89.5%) and tetracycline (89.5%). E. coli were found resistant against all the classes of antimicrobials used at the farm level. Tetracycline resistance gene (tetA) was detected in 100% of the tetracycline resistant E. coli and blaTEM-1 was present in 38.9% of the E. coli isolates. Findings of this study indicate a potential threat of developing antimicrobial resistance in commensal E. coli and their association with clinical mastitis. Occurrence of multidrug resistant E. coli might be responsible for the failure of antibiotic therapies in clinical mastitis as well as pose potential threat of transmitting and development of antibiotic resistance in human.

Abstract LB196: Comparison of whole exome sequencing and targeted sequencing in patients with advanced bladder cancer

Abstract Background: Next generation sequencing (NGS) to identify genomic alterations (GAs) in tumor samples are increasingly used in clinical practice. Targeted sequencing (TS) or whole exome sequencing (WES) are used by various commercially available platforms. Differing methods often result in poor inter-reproducibility, even when testing the same tissue. Bladder cancer has substantial genetic heterogeneity as compared to many other malignancies. The purpose of this study is to compare WES and TS in bladder cancer. Methods: Patients with advanced bladder cancer who had undergone tissue WES (GEM ExTra®) or TS (FoundationOne CDx® [FMI]) in the course of routine clinical care were assessed. Genes with at least one detected GA were selected, with others excluded. All detected GAs were classified as being detected on TS only, WES only, concordant (detected on both platforms), or partially concordant (different GAs detected within the same gene). Variants of uncertain significance were excluded for this study. Results: In our series, 25 patients had WES and 42 patients had TS - 14 patients (21%) had both tests performed. A total of 88 genes were detected to have at least one GA across all samples. Of all detected GAs, 66/162 (41%) were concordant, with 45/162 (28%) and 51/162 (31%) of GAs detected exclusively by WES and TS, respectively. The genes with the highest proportion of concordance when detected, were TERT (70%), TP53 (85%) and ARIDA1A (50%). SOX4 and E2F3 were detected on WES only, while RBM and CHEK2 were detected on TS only. Notably, six TP53 GAs were detected exclusively on TS. TP53, TERT, MLL2/KMT2D, ERBB2 were each partially concordant on one occasion. The difference in the rate of concordant GAs in samples from the same collection site and date (41%), as compared to those in which both differed (35%) were not statistically significant (p=0.54). Conclusions: Although the majority of GAs identified were concordant, each platform exclusively identified GAs with varying clinical significance. SOX4 and E2F3 mutation, shown to correlate with invasive potential in bladder cancer, were detected exclusively on WES. In contrast, RBM and CHEK2 (of known significance in melanoma and breast cancer) were found exclusively on TS. Larger series are needed to further establish the concordance of these approaches. Citation Format: Neal Shiv Chawla, Zeynep Zengin, Ameish Govindarajan, Sumanta K. Pal. Comparison of whole exome sequencing and targeted sequencing in patients with advanced bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB196.

Abstract 244: A computation method for noise reduction based on ultra-deep targeted sequencing data

Abstract Detection of circulating tumor DNA is a promising approach for the diagnosis and monitor of solid tumors. However, sensitive detection of ctDNA remains challenging when minor allele frequency (MAF) is ultra-low. Numerous noise reduction data algorithms have been developed to reduce next-generation sequencing (NGS) errors, but variant callers that can take advantage of ultra-deep sequencing data are lacking. Here we develop a bin-based genotyping (BinGo) method for canceling sequencing noise using ultra-deep (&amp;gt; 100,000 X depth) targeted sequencing data. A mutation reference sample with 44 mutations was diluted to different MAFs at 1% and 0.5%. One blood sample for proficiency test was included. We constructed targeted sequencing libraries by anchored multiplex enrichment for a panel of targets that cover the 44 mutations, and sequenced to a very high depth (Mean X ranged: 71K to 350K).After raw NGS reads were mapped by BWA MEM, BinGo tags unique molecular index (UMI) to each alignment (BAM) to preserve alignment signal of all bases in all raw reads. BinGo then bins all reads with the same UMI into one bin, generating millions of bins. Millions of parallel Samtools mpileup was enabled by the GNU parallel tool. Typical mpileup analyses use the human genome (requires ~4GB RAM) as a reference, making millions of such analyses essentially impossible in a regular computing server. To overcome this hurdle, BinGo uses tailor-made reference sequences comprising the targets only (&amp;lt; 100KB), drastically reduces computation memory consumption and enables millions of mpileup analyses. BinGo recompiles Samtools to increase its default max depth from 8,000 to 8,000,000 to accommodate the ultra-deep data. Variants derived from the same bin are effectively phased, thereby enabling calling of di-nuclei mutations such as BRAF V600K (GT&amp;gt;TT) and V600R (GT&amp;gt;AG). This binning also allows for sensitive calling of insertions and deletions even down to a single read. To profile noise patterns, BinGo uses Samtools tview to acquire every base in every alignment position of the target sequences in all raw reads. Finally, BinGo uses multiple statistical testing to call variants, including calling nucleotide changes over the background noise model that is defined by nucleotide change and sequence context; filtering out strand imbalance likely due to DNA template damage, and a low number of supporting ligation starting sites. With 10 ng DNA as input and at an expected MAF of 0.5%, BinGo demonstrates a 100% (44/44) analytical sensitivity and 100% specificity. When using 1 ng DNA as input and at an expected MAF of 0.5%, only one mutation was missed, leading to a sensitivity of 97.73% (43/44) and 100% specificity. Furthermore, BinGo detected in the blood DNA sample a 15-bp EGFR exon 19 deletion at 0.05%.Bingo integrates UMI-bin derived variants and raw sequencing background noise modeling, enabling analysis of ultra-deep targeted NGS data for rare variant detection. Citation Format: Weiwei Bian, Firaol Kebede, Zongli Zheng. A computation method for noise reduction based on ultra-deep targeted sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 244.

Abstract 3011: Cancer of Unknown Primary: novel therapeutic opportunities from patient-derived cell cultures and in vivo models

Abstract Patients with occult primary cancer or cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies, which are typically tumor-type oriented. Moreover, experimental in-vitro and in-vivo models summing up CUP features and heterogeneity are currently unavailable. We derived and expanded two CUP cell lines, and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines, one spontaneously growing as organoid, and PDXs were subjected to histological, immune-phenotypical, molecular and genomic (CNV and target sequencing) characterization to show their ability to recapitulate the original tumor. MicroRNA profile and genomic information were used to identify a possible primary site and patient-specific therapeutic approaches. Data on CUP most frequently altered genes were used to design a custom CUP-specific, 92-gene panel for target NGS analysis with Sure Select XS technology (Agilent Technology). Drug-screening assays were performed in vitro and in vivo on two models targeting the most promising actionable pathways. We obtained unexpected and promising results on drug combinations efficacy. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Claudia Fumarola, Elisa Porcellini, Stefano Cairo, Giulia Gallerani, Francesco Gelsomino, Irene Salamon, Maria Naddeo, Andrea Ardizzoni, Andrea Cavazzoni, Manuela Ferracin. Cancer of Unknown Primary: novel therapeutic opportunities from patient-derived cell cultures and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3011.