Generation Sequencing Methods Research Articles

O-281 Characterization of Vaginal microbiota and reproductive outcomes associated with assisted reproductive technologies through next-generation sequencing, from Indian population

Abstract Study question Is there any association between the composition of the vaginal microbiota and the reproductive outcomes in infertile women’s undergoing for assisted reproductive treatments (ART)? Summary answer Higher microbial abundance in non-pregnant patients compared to pregnant patients. These pathogens could be used as a possible marker for a higher IVF failure rate. What is known already Various next- generation sequencing methods have been used to study bacterial populations in infertile Women. Vaginal microbiota could have an influence on success rates of fertility treatments. The bacterial microbiota varies by the population. There is no direct evidence on this subject to guide clinical decision making or its influence on the hierarchy of fertility treatments. While the presence of pathogenic bacteria non-Lactobacilli dominated microbiota is associated with poor reproductive outcomes in assisted reproductive treatments (ART). Additional studies, particularly those using metagenomics approaches, may be useful. Study design, size, duration A total of 240 infertile women’s were included in the study. The study population consists of patients attending to the ART unit of a tertiary medical center Udaipur, India, between November 2021 to October 2023. Depending on the biochemical pregnancy results, they were divided into two groups. Group 1 (n = 128) was pregnancy (P), and Group 2 (n = 112) non- pregnancy (NP) group, Patients were aged between (20-45 years). The ethical approval was obtained from the institute. Participants/materials, setting, methods DNA was extracted from vaginal swab samples collected at the time of oocyte pick up using the commercially available ( DNeasy Power Soil kit). The Vaginal microbiota of samples were analyzed using 16S ribosomal RNA gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity and taxonomic characterization were compared for microbial composition. The findings of the principle coordinate analysis were shown using R version 4.0. Main results and the role of chance Our results show that higher Shannon and Simpson index were obtained in the non- pregnancy (NP) group compared to the pregnancy (P) group. In addition, the partial least squares discriminant analysis (PLS-DA) revealed a difference in microbial composition between pregnancies (P) and non- pregnancy (NP) groups, as well as a higher microbial abundance in non-pregnant patients compared to pregnant patients. At the phylum level Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, and Bacteroidetes are the most common predominant in the pregnancy (P) group (80.77, 9.52, 9.18, 0.04, and 0.40%, respectively) and the non- pregnancy (NP) group (72, 61, 12.41, 8.01, and 3.75%). A higher abundance of Firmicutes (80.77 vs. 72.61%) and Proteobacteria (9.18 vs. 8.01%), and a lower abundance of Actinobacteria (9.52 vs. 12.41%, p = 0.03), Fusobacteria (0.04 vs. 8.01%, p = 0.02), and Bacteroidetes (0.40 vs. 3.75%, p = 0.03) were observed in pregnancy (P) group. At the genus level, pregnancy group (P) increased the abundance of probiotics lactic acid bacteria (74.61 vs 63.09%) and decreased the abundance of the pathogen Gardnerella (6.03 vs 7.24%, p = 0.03), Atopobium (0.84 vs 4.14%, p = 0.02), Sneathia (0.03 vs. 3.75%, p = 0.02), and Prevotella (0.38 vs. 3, 02%, p = 0.04). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in female reproductive tracts samples in connection to infertility and live birth rate (LBR) outcomes. Wider implications of the findings Vaginal microbiota composition associated to different clinical outcomes in patients undergoing ART might have profound implications. This may lead to failed fertility treatments and reduced live birth rate (LBR). More studies are needed to clarify the relative effectiveness of treatments for infertility. Trial registration number NA

Electronic Medical Record-Based Nudge Intervention to Increase Comprehensive Molecular Genotyping in Patients With Metastatic Non-Small Cell Lung Cancer: Results From a Prospective Clinical Trial.

Less than half of the patients with newly diagnosed metastatic non-small cell lung cancer (NSCLC) undergo comprehensive molecular testing. We designed an electronic medical record (EMR)-based "nudge intervention" to prompt plasma-based molecular testing at the time of initial medical oncology consultation. A nonrandomized prospective trial was conducted at the University of Pennsylvania's academic practice and two affiliated community practices. Molecular genotyping was performed by tissue- and/or plasma-based next generation sequencing methods. Comprehensive testing was defined as testing for EGFR, ALK, BRAF, ROS1, MET, RET, KRAS, and NTRK. Guideline-concordant treatment was defined as the use of the appropriate first-line (1L) therapy as per the National Comprehensive Cancer Network (NCCN) guidelines. Proportion of patients with comprehensive molecular genotyping results available at any time, molecular results available before 1L therapy, and guideline-concordant 1L treatment were compared between the preintervention and postintervention cohorts using Fisher's exact test or Pearson's chi-squared test. Five hundred and thirty-three patients were included, 376 in the preintervention cohort and 157 in the postintervention cohort. After implementation of the EMR-based nudge, a higher proportion of patients underwent comprehensive molecular testing in the postintervention versus the preintervention cohort (100% v 88%, P = <.001), had results of comprehensive molecular testing available before initiating 1L treatment (97.3% v 91.6%, P = .026), and received NCCN guideline-concordant care (89.8% v 78.2%, P = .035). Across three practice sites in a large health system, implementation of a provider team-focused EMR-based nudge intervention was feasible, and led to a higher number of patients with NSCLC undergoing comprehensive molecular genotyping. These findings demonstrate that behavioral nudges can promote molecular testing and should be studied further as a tool to improve guideline-concordant care in both community and academic sites.

#234 Clinical spectrum and genetic variants of Alport syndrome and thin basement membrane nephropathy—single center experience

Abstract Background and aims Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetically heterogeneous kidney diseases that manifest with kidney involvement, AS also with hearing and visual impairment. Electron microscopic examination shows characteristic ultrastructural changes in the glomerular basement membrane (GBM) in AS and TBMN, but cannot reliably distinguish between the two entities. Therefore, molecular genetic studies and the identification of individual pathogenic variants in the target genes are crucial to confirm the diagnosis. The aim of our study is to determine the significance of the genetic variants associated with the ultrastructural changes of GBM found in the renal biopsy. Method We retrospectively analyzed records of patients from the University Medical Centre Ljubljana in which ultrastructural GBM changes characteristic for AS and TBMN were found in the renal biopsy, including their clinical, laboratory and histological features. Molecular genetic investigations were made using the new generation sequencing method (NGS) to determine germline pathogenic variants in genes directly related to AS and TBMN (COL4A3, COL4A4 and COL4A5) and in genes that are important in syndromes with a related clinical picture (COL4A6, CD151, LMX1B, MYH9, FN1 and MYO1E). The pathogenicity of variants was determined according to the Clinical Molecular Genetic Society best practice guidelines. Results The study included 73 patients with ultrastructurally confirmed thin GBM. Indications for kidney biopsy were chronic nephritis syndrome in 33 patients (45%), acute worsening of renal function in 16 (22%), nephrotic syndrome in 9 (12%), nephrotic proteinuria and chronic kidney disease in 6 (8%), isolated proteinuria in 5 (7%) and suspected vasculitis in 4 patients (6%). At the time of kidney biopsy average age was 50 ± 16 years, average creatinine 112 ± 70 µmol/L and average estimated glomerular filtration rate (eGFR) 65 ± 24 ml/min/1.73 m2, 7 patients had eGFR below 30 ml/min/1.73 m2. Average daily proteinuria was 2.4 ± 3.3 gr and 88% of patients had hematuria. 38% of them have hearing impairment and 43% had positive familiar history of kidney disease. We recorded 33 patients (45%) with genetic variants associated with AS or TBMN: 22 patients (67%) with pathogenic variants, 2 patients (6%) with likely pathogenic and 9 patients (27%) with variants of uncertain significance. The presence of a positive genetic analysis was associated with a higher level of hematuria (p = 0.003), a lower level of proteinuria (p = 0.004) and a positive family history of kidney disease (p &amp;lt; 0.001). It was also associated with hearing loss, but the association was not statistically significant (p = 0.099). Patients with genetic variants had similar eGFR to those with negative genetics (63.9 ml/min/1.73 m2 (58.3-69.4) vs 66.2 ml/min/1.73 m2 (60.0-72.3)), p = 0.698. We also found no differences in GBM thickness in patients without compared to those with identified gene variants (213 nm (160-315) vs. 209 nm (140-410)), p = 0.761. 32 patients (44%) had only focal chronic changes and ultrastructural changes of thin GBM present on kidney biopsy and 41 patients (56%) had also been diagnosed with other renal pathology. When comparing these two groups, patients without associated pathology had lower daily proteinuria (p = 0.048), higher level of hematuria (p = 0.021), frequent hearing impairment (p = 0.008) and also positive genetics analysis (p = 0.016). Conclusion We have shown that in patients with proven ultrastructural alterations in GBM, the presence of so far recognized genetic variants associated with AS and TBMN is 45%, and corresponds to the frequency of other phenotypic characteristics of AS, including hearing impairment and positive family history. In the future, additional clinical tools will be required to accurately determine the role of coexistent thin GBM on the prognosis of patients with other relevant kidney pathology and other genetic tools beyond NGS should be employed to explore other pathologic genetic variants associated with the AS spectrum.

Exploration of the polypropylene degrading bacteria candidates from the passive zone of the Supit Urang landfill in Malang city by using the next generation sequencing (NGS) method

Exploration of the polypropylene degrading bacteria candidates from the passive zone of the Supit Urang landfill in Malang city by using the next generation sequencing (NGS) method

Abstract 7024: Development of quality control methods for reliable NGS measurement of methylation in cfDNA

Abstract Background and Purpose: Reliable measurement of circulating free genomic DNA (cfDNA) methylation patterns promises a means for cancer early detection as well as treatment response. Use of Next Generation Sequencing (NGS) methods to measure the cfDNA methylome across a large number of potentially methylated CpG sites may enable earlier detection by overcoming stochastic sampling associated with the small specimens obtainable by a blood draw. Wide-spread adoption of these methods for clinical diagnostic applications will be facilitated through improved quality controls that increase reliability. The goal of this study is to assess the performance of the SNAQ-SEQ™ method as a means to establish a limit of detection (LOD) for measurement of methylation at each targeted CpG site. For this purpose, we used test materials that were developed by the National Institute for Standards and Technology (NIST) to mimic methylated cfDNA. Methods: NIST test materials were formulated by physically shearing cell line gDNA then subjecting it to size separation to mimic a size distribution similar to that observed in cfDNA, then mixing sheared DNA in its “native” state of methylation with sheared DNA that was in vitro methylated. Non-sheared NIST test materials also were assessed in these studies. Synthetic spike-in internal standards (IS) were combined with NIST test materials and the combined samples were bisulfite converted, then subjected to PCR-amplicon library preparation. Libraries were sequenced on an Illumina MiSeq instrument and bioinformatic analysis was conducted using the Qiagen CLC Workbench. Results and Conclusions: In initial testing of an off-the-shelf IS for a region of the SOX2 gene, average methylation (preserved C) across 14 targeted CpG sites was 16.4% (0.5%-50%) in the “native” NIST non-sheared sample. This was consistent with the 11.6% methylation across all of SOX2 measured by the NIST. In contrast, in IS corresponding to the targeted SOX2 region the average preserved C was 0.32% (0.03%-0.5%). Use of synthetic internal standards enabled determination of LOD for each individual methylated site, controlling for all known sources of technical error, including inefficient bisulfite conversion, nucleotide substitution error, and alignment error. Studies are now underway that use the NIST test materials to measure methylation at five targets known to be altered in cfDNA from lung cancer subjects, SOX2, CDO1, TAC1, SOX17, and HOXA7. Citation Format: James C. Willey, Erin L. Crawford, Daniel J. Craig, Thomas M. Morrison, Hua-Jun He, James G. Herman. Development of quality control methods for reliable NGS measurement of methylation in cfDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7024.

Association of candidate alterations with primary resistance to KRAS G12D targeting in colorectal cancer.

170 Background: KRAS-mutant colorectal cancer (CRC) has a worse prognosis and greater resistance to therapy, attracting tremendous efforts to develop both allele-specific and pan-KRAS targeting agents including KRAS G12C and G12D inhibitors. CRC has demonstrated both primary and acquired resistance to KRAS G12C inhibitors, which can be better evaluated by circulating tumor DNA (ctDNA)-based next generation sequencing (NGS) methods without tissue sampling bias. Here, we utilized clinical ctDNA data to delineate the landscape of candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Methods: Samples from advanced CRC patients with Guardant360 data in 2021 were categorized into a KRAS G12D group and comparator groups of KRAS G12C and KRAS not detected (ND) according to KRAS mutation status. Demographics, MSI-H status, and blood tumor mutational burden (bTMB) were summarized and compared between groups using t-test for continuous variables and Fisher’s exact test for categorical variables. Co-occurrence patterns of alterations between KRAS and other genes were assessed. Primary resistance mutations were inferred by acquired resistance findings to KRAS G12C inhibitors reported by Awad et al. N Engl J Med. 2021 and Tanaka et al. Cancer Discov. 2021. Results: Among approximately 2500 samples, 16.8% had KRAS G12D, the most common KRAS mutation in CRC. The mean allele frequency of KRAS G12D (11.4%) is comparable to that of KRAS G12C (10.9%). Mean age (61.6 years), MSI-H frequency (3.8%), and mean bTMB (14.2 Mut/Mb) of KRAS G12D group is comparable to those of KRAS ND group. This contrasts with KRAS G12C with significantly lower mean age (60.2 years, p=0.01), lower MSI-H frequency (0.56%, p=0.03), and higher mean bTMB (16.3 Mut/Mb, p=0.02) compared to KRAS ND. Both KRAS G12D and G12C significantly co-occur with APC, PIK3CA, and SMAD4 alterations. FBXW7, AR, and KEAP1 alterations were unique co-occurrences to KRAS G12D, while KIT was unique to KRAS G12C. Candidate alterations associated with primary resistance in KRAS G12D CRC included simultaneously present mutations of KRAS Q61H (14.1%), G12V (6.4%), G12C (5.9%), G13D (3.6%), BRAF V600E (2.7%) and others, amplification of MET (2%), MYC (1.6%), and KRAS (1.4%), and FGFR3-TACC3 translocation (Table). Conclusions: KRAS G12D is associated with unique co-occurring molecular alterations compared to KRAS G12C in CRC. ctDNA-based NGS platforms can survey candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Further validation in preclinical models is needed. [Table: see text]

Comparison of Sinus Flora Using Various Next-Generation Sequencing Techniques Versus Standard Culturing

Background:Next generation sequencing methods are being developed to help diagnose infectious diseases at the point of care and to help resolve discrepant results. These new methods hope to replace longer, more tedious sequencing methods such as Sanger/shotgun sequencing and less sensitive culture results to provide a clinical diagnosis and start appropriate treatment sooner. In lieu of dated techniques, with next generation sequencing the physician could analyze samples in their clinic with relative ease and receive a sensitive diagnosis in a fraction of the time.&#x0D; Objective:To compare percent agreement between standard culturing results of swabbed sinus samples on blood and chocolate agar to BIOFIRE® FILMARRAY® results, and use Oxford Nanopore and 16s illumina sequencing results to resolve discrepancies.&#x0D; Study design:Swabs were taken of the nasal sinuses of 21 patients and flash frozen. Some of these swabs (15/21) were sequenced with 16s illumina sequencing. All these swabs were put into a saline solution and plated on blood and chocolate agar plates as a 1:100 loop dipped into the solution and as the original swab. These solutions were sequenced through the BIOFIRE® FILMARRAY® Torch System on a pneumonia and blood culture (BCID2) panel and through the Oxford Nanopore® MinION Mk1C® system.&#x0D; Results:The pneumonia panel had 36% agreement with blood agar and 52% agreement with chocolate agar plates. Using the top 3 genus results, either Nanopore or 16s illumina sequencing resolved 92% of discrepancies between the pneumonia panel and blood agar and 90% of discrepancies between the pneumonia panel and chocolate agar plates. The blood culture panel had 43% agreement with blood agar and 57% agreement with chocolate agar plates. Nanopore or 16s illumina sequencing resolved 92% of discrepancies between the blood panel and blood agar and 89% of discrepancies between the blood panel and chocolate agar.

Predmoter-cross-species prediction of plant promoter and enhancer regions.

Identifying cis-regulatory elements (CREs) is crucial for analyzing gene regulatory networks. Next generation sequencing methods were developed to identify CREs but represent a considerable expenditure for targeted analysis of few genomic loci. Thus, predicting the outputs of these methods would significantly cut costs and time investment. We present Predmoter, a deep neural network that predicts base-wise Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) and histone Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) read coverage for plant genomes. Predmoter uses only the DNA sequence as input. We trained our final model on 21 species for 13 of which ATAC-seq data and for 17 of which ChIP-seq data was publicly available. We evaluated our models on Arabidopsis thaliana and Oryza sativa. Our best models showed accurate predictions in peak position and pattern for ATAC- and histone ChIP-seq. Annotating putatively accessible chromatin regions provides valuable input for the identification of CREs. In conjunction with other in silico data, this can significantly reduce the search space for experimentally verifiable DNA-protein interaction pairs. The source code for Predmoter is available at: https://github.com/weberlab-hhu/Predmoter. Predmoter takes a fasta file as input and outputs h5, and optionally bigWig and bedGraph files.

Bacteria Analysis in the Ciwalen River Garut using Next Generation Sequencing of 16S Ribosomal RNA

&#x0D; &#x0D; &#x0D; The Ciwalen River flows through Garut city, West Java, Indonesia. This river is bordered by hundreds of leather tanning enterprises and community toilet waste. The purpose of this research is to analyse bacteria in Ciwalen river water using the Next Generation Sequencing (NGS) method for the 16s ribosomal RNA gene using V1-V9 primer pairs, which will amplify the entire length of the rRNA gene and allow for species identification. The data revealed that there were 104,000 reads, 150,232,539 bases, and 81,761 bacterial species. Proteiniclasticum ruminis, Fusibacter ferrireducens, Fusibacter fontis, Fusibacter tunisiensis, Paludibacter propionicigenes, Arcobacter cloacae, Alishewanella jeotgali, Thiovirga sulfuroxydans, Pseudomonas fluvialis, and Thauera mechernichensis are the main bacterial species. In this study, another bacteria, Klebsiella pneumoniae, was also found which is known to degrade heavy metals, chromium, timbal &amp; cadmium (Cr, Pb and Cd). The profile of bacteria, as well as the presentation of their existence in this water sample, demonstrates the appropriateness of heavy metals from leather tannery waste in the Ciwalen river.&#x0D; &#x0D; &#x0D;

Exploration The Candidates of Xenobiotic Degrading Indigenous Bacteria from Probolinggo City Landfill by Using Next Generation Sequencing (NGS)

Soil bacteria from tropical environments play a significant role in resolving various environmental issues, including biodegradation. Exploratory research on biodiversity is crucial to develop and harness the potential of different types of soil bacteria that are highly abundant. The bacterial diversity in landfills is typically high due to the decomposition of organic and inorganic waste, creating a favorable medium for the growth and development of soil bacteria. This study aims to assess the candidates of xenobiotic degrading indigenous bacteria from the Probolinggo City landfill using Next Generation Sequencing (NGS) method. The research stages include: 1) sampling, 2) isolation of genomic DNA from samples using the ZymoBIOMICS DNA MiniPrep Kit from Zymo Research, 3) amplification of isolated DNA with primers 16S 27F – 1429R, 4) sequencing the results of DNA amplification with NGS, 5) downstream analysis of the results using software Pavian Krona Tools, and 6) narrative analysis review to identify the candidates of xenobiotic degrading indigenous bacteria. The results show that soil samples from the Probolinggo City landfill exhibited a high diversity of bacterial communities. Based on NGS analysis, 2400 bacterial species were identified, comprising 56 genera, 17 orders, 4 classes, and 4 phyla, with respective abundances of Proteobacteria (70%), Firmicutes (15%), Planctomycetes (2%), and Cyanobacteria (0,3%). Based on the narrative analysis review, several bacteria in the Probolinggo City landfill exhibited potential as: 1) polypropylene-degrading bacteria, including Bacillus cereus, B. licheniformis and B. thuringiensis. 2) styrofoam degrading bacteria, namely Bacillus amyloliquefaciens, Bacillus cereus, Bacillus firmus and Pseudomonas aeruginosa. 3) total ammonia nitrogen (TAN) reducing bacteria, including Bacillus megaterium. 4) pesticide degrading bacteria Profenofos and Chlorantraniliprole, including Bacillus stearothermophilus. and 5) tannic acid degrading bacteria, including Pantoea dispersa. These results indicate that the Probolinggo City landfill is a good habitat for various xenobiotic-degrading bacteria, then the isolation of specific bacteria can be designed using an appropriate selective medium.

Efficient extraction of adventitious virus nucleic acid using commercially available methods

An essential step in pharmaceutical product development is screening for contamination with adventitious agents, and there is desire to develop highly sensitive assays to detect adventitious viral nucleic acid. This study sought to examine the nucleic acid extraction efficiency of three viral candidates in relevant background matrices using four different extraction methods. Three model adventitious viruses, Minute virus of Mice, Porcine Circovirus, and Feline Leukemia Virus, were diluted within a variety of background matrices relevant to pharmaceutical production methods. Upon extraction, the nucleic acid was quantified using droplet digital PCR methods. Four nucleic acid extraction methods were assessed, including commercially available kits and manual extraction methods. Each method recovered nucleic acid post-extraction for each of the model viruses within the tested background matrices. The silica-column based method recovered a greater amount of viral nucleic acid, compared to the other methods tested. Similar trends were observed when model virus was diluted in bioreactor supernatant, which replicates industry testing conditions and provides details on which extraction methods might be used in Next Generation Sequencing and PCR methods for detecting contamination within pharmaceutical products.

Молекулярное разнообразие рабдомиосаркомы. Применение метода высокопроизводительного секвенирования

Поиск новых подходов для индивидуализации терапии пациентов детского возраста с рабдомиосаркомами является актуальным направлением современных научно-практических исследований. Молекулярно-генетические особенности опухоли могут быть эффективно использованы для более точного прогнозирования клинического исхода заболевания, генетически направленной терапии, а также посттерапевтического мониторинга качества ремиссии. Не вызывает сомнений, что в основе успешного лечения и контроля агрессивности клинического течения болезни лежит детальное изучение и глубокое понимание механизмов регуляции опухолевой прогрессии, что определяет актуальность этого направления исследований. В настоящей работе представляются результаты проведенного высокопроизводительного секвенирования для образцов опухолевой ткани 21 пациента с установленным диагнозом РМС (66,6% эмбрионального подтипа РМС и 33,4% альвеолярного подтипа РМС). В ходе анализа полученных данных было обнаружено 85 мутаций (однонуклеотидных замен, инсерций, делеций) в 170 различных генах. Наиболее часто мутации были обнаружены в генах EGFR (12/85; 14,1%), NRAS (8/85; 9,4%), BRCA (7/85; 8,2%), MYC (6/85; 7%), CHEK (6/85; 7%) и NF1 (5/85; 5,8%), MYCN (4/85; 4,7%). Находкой данного исследования является обнаружение новой транслокации – NSD1/FGFR4, которая на момент анализа актуальных литературных данных не описана при РМС, но является патогенной драйверной молекулярной поломкой и имеет онкогенный потенциал у генов-партнеров при другой онкопатологии. The search for new approaches to the individualization of therapy for children with rhabdomyosarcoma is an actual direction of modern scientific and practical research. Molecular and genetic features of the tumor can be effectively used for more accurate prediction of the clinical outcome of the disease, genetically directed therapy, as well as post-therapeutic monitoring of the quality of remission. There is no doubt that the basis of successful treatment and control of the aggressiveness of the clinical course of the disease lies in a detailed study and deep understanding of the mechanisms of tumor progression regulation, which determines the relevance of this direction of research. This paper presents the results of high-throughput sequencing of tumor tissue samples from 21 patients with an established diagnosis of RMS (66.6% embryonic subtype RMS and 33.4% alveolar subtype RMS). During the analysis of the obtained data, 85 mutations (single nucleotide substitutions, insertions, deletions) were found in 170 different genes. The most frequent mutations were detected in the genes EGFR (12/85; 14.1%), NRAS (8/85; 9.4%), BRCA (7/85; 8.2%), MYC (6/85; 7 %), CHEK (6/85; 7%), and NF1 (5/85; 5.8%), MYCN (4/85; 4.7%). The finding of this study is the discovery of a new translocation – NSD1/ FGFR4, which at the time of analysis of current literature data has not been described in RMS, but is a pathogenic driving molecular breakdown and has oncogenic potential in partner genes in the second oncology.

Wound healing of experimental equine skin wounds and concurrent microbiota in wound dressings following topical propylene glycol gel treatment.

Topical wound treatments rely on carrier formulations with little to no biological impact. The potential for a common vehicle, a propylene glycol (PG) gel, to affect wound healing measures including microbiota is not known. Microbiome characterization, based on next generation sequencing methods is typically performed on tissue or directly obtained wound fluid samples. The utility for primary wound dressings to characterize equine wound microbiota in the context of topical treatments is currently unknown. This investigation reports the topical effect of an 80% PG based gel on wound healing and microbiota in wound dressings. Experiments were performed in six mature horses utilizing a surgical, distal limb wound model, histology of sequential wound biopsies, photographic wound measurements and microbiota profiling via 16s rRNA sequencing of wound dressing samples. Experimental wounds were surveyed for 42 days and either treated (Day 7, 14, 21 and 28; at 0.03 ml/cm2) or unexposed to the PG gel. Wound surface area, relative and absolute microbial abundances, diversity indices and histologic parameters were analyzed in the context of the experimental group (treatment; control) using qualitative or quantitative methods depending on data characteristics. Compared to controls, treatment slowed the wound healing rate (17.17 ± 4.27 vs. 18.56 ± 6.3 mm2/day), delayed the temporal decline of polymorphonucleated cells in wound beds and operational taxonomic units (OTU) in wound dressings and lowered alpha-diversity indices for microbiota in primary wound dressing. Relative abundances of OTUs were in line with those previously reported for equine wounds. Clinical outcomes 42 days post wounding were considered similar irrespective of PG gel exposure. Results highlight the potential for vehicle exposure to alter relevant wound outcome measures, imposing the need for stringent experimental control measures. Primary wound dressings may represent an alternate sample source for characterization of the wound microbiome alleviating the need for additional interventions. Further studies are warranted to contrast the microbiome in wound dressings against that present on wound surfaces to conclude on the validity of this approach.

Mutation analysis of the TERT gene in ovarian cancer patients of the Turkish population by next generation sequencing method.

Ovarian cancer (OC) ranks seventh among malignant tumors worldwide. As one of the most common gynecological malignancies, ovarian cancer has the second-highest mortality rate, after cervical and uterine cancer. Next-Generation Sequencing (NGS) technology has enhanced multi-gene panel analysis and its clinical utility for identifying cancer-causing gene mutations. This study aimed to determine the presence of significant and nonsense mutations in telomerase reverse transcriptase (TERT), alpha-thalassemia/mental retardation, X-linked (ATRX), O-6-methylguanine-DNA methyltransferase (MGMT), and isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) genes using the Next-Generation Sequencing (NGS) method. A cohort of 33 patients diagnosed with ovarian cancer was included in this investigation, and peripheral blood samples were collected from all participants. Significant and nonsense mutations in TERT, ATRX, MGMT, IDH1, and IDH2 genes were detected using the Next-Generation Sequencing method. Bioinformatics analysis was conducted using the QIAGEN Clinical Insight system. Twenty-four patients exhibited seven different TERT mutations, occurring in both exonic and intronic regions. One patient displayed a c.699-3delC deletion in the intronic region of the IDH1 gene, and the c.532G > A (p.V178I) mutation observed in three patients was assessed as potentially harmful. Additionally, novel mutations c.881A > G and c.995A > G were observed in the ATRX gene. The heterozygous novel mutation identified in the ATRX gene was confirmed through Sanger sequencing. These mutations were not previously associated with ovarian cancer and are considered novel candidate markers for ovarian cancer susceptibility. Confirmation of these results through larger cohort studies or functional investigations will contribute to a better understanding of the molecular mechanisms underlying ovarian cancer.

Diversity analysis of southern African Artemisia afra using a single nucleotide polymorphism derived from diversity arrays

Artemisia afra is an aromatic shrub used as a herbal remedy in southern Africa for various disorders, including coughs, colds, influenza, and, most notably, for the prophylaxis and treatment of Plasmodium falciparum infections. Modern high-throughput genotyping by sequencing, which combines next generation sequencing and diversity array technology (DArTseq) complexity reduction methods, allows for the generation of high-quality whole-genome profiles without any previous organismal genetic information that may hamper the diversity analyses. This study investigated the feasibility of incorporating DArTseq-derived single-nucleotide polymorphisms (SNPs) and bioactive compounds into a geographic information system to predict the spatial distribution of A. afra gene diversity in selected southern African environs. DNA extraction, sequencing, and SNP calling mineral analyses (using R Studio) were performed on a total of 80 genotypes collected around Roma and Mohale’s Hoek, Lesotho, and Wepener and Hobhouse, South Africa. The unweighted pair group method with arithmetic mean (UPGMA) and neighbour-joining dendrograms revealed three clusters of variation among genotypes; however, geographic distribution did not influence A. afra genetic diversity. The mean heterozygosity among genotypes was 7.7%. Genotypes collected in Roma and Mohale’s Hoek exhibited high levels of genetic diversity. Local A. afra genetic diversity was relatively low, indicating uniformity of subspecies per locality. The diversity among genotypes indicated that A. afra SNPs may occur despite the location. Given that genetic diversity enhances crop performance, our findings can be useful in germplasm management, industries producing by-products of A. afra, and plant breeding programs. For the long-term conservation of medicinal plants in South Africa, significant sampling and regional data are required. In addition to evaluating qualitative and quantitative assessments of phytochemicals, it is necessary to evaluate foliar mineral composition, as well as the agronomic and morphological characteristics of collected specimens from studied locations.

SYNE1 gene novel variant is associated with myocardial infarction in young people with a family history of premature atherosclerosis

Abstract Introduction Genetic background is thought to be responsible for a family occurred premature atherosclerosis, including myocardial infarction (MI). Purpose The aim of the study was to investigate the role of genetic variants in young patients (aged &amp;lt;50 years) with MI and family history of premature atherosclerosis. Methods The studied group consisted of 70 patients aged 26-49 (mean 43.1, SD ±4.3), 17 women and 53 men, with MI and with family history of premature atherosclerosis, defined as MI or ischaemic stroke in first-degree relatives at age &amp;lt;65 years in women or &amp;lt;55 years in men. The mean BMI in the studied group was 28.8 kg/m2 (SD ±3.5), total cholesterol level 202.3 mg/dl (SD ±3.5), HDL 40.6 mg/dl (SD ±9.6), LDL 124.0 mg/dl (SD ±28.9), triglycerides 164.4 mg/dl (SD ±63.5), glucose 108.3 mg/dl (SD ±19.9) and creatinine 0.9 mg/dl (SD ±0.2). Regarding other risk factors 63 patients (90%) were smokers (mean 24.1 pack-years, SD ±13.0), 44 (62.8%) had hypertension, 11 (15.7%) had diabetes mellitus, and 8 (11.4%) had depression. All patients had diagnosed MI including MI with ST elevation (STEMI) in 18 cases (25.7%) and MI without ST elevation (NSTEMI) in 52 cases (74.3%). All patients, except 2 (no consent), had performed coronary angiography revealing 1-vessel coronary artery disease (VCAD) in 33 patients (47.1%), 2-VCAD in 17 patients (24.3%), and 3-VCAD in 20 patients (28.6%). The mean left ventricular ejection fraction (LVEF) was 51.8% (SD ±8.0). The total DNA has been extracted from whole peripheral blood samples. The targeted enrichment library was prepared and analyzed using New Generation Sequencing method on Hiseq4000, 2x150 cycles. Statistical analyses were performed using the R software package (www.r-project.org/). The results of sequencing were compared to data from the reference control population consisting of 597 healthy people (418 women, 179 men) aged 18-83 (mean 40.5, SD ± 12,4) as a whole and after matching with a studied group by age and gender in a proportion 1:3 (210 people, 51 women and 159 men, aged 18-77, mean 42.1, SD ±10.6) using Propensity Score Matching. Risks associated with detected variants were evaluated using Fisher’s exact test based on the allelic frequencies of variants in both groups. Results SYNE1 gene variant rs36215567 (NM_182961.4: c.20396+22A&amp;gt;G) has been detected in statistically significant higher incidence in the studied group in comparison to the control population (p=0.005) with OR=4,80 (95%CI 1,43-14,45) as well as when compared to the control population matched by age and gender (p=0.004) OR=9,31 (95%CI 1,64-95,41). There were no statistically significant differences in the incidence of familial hypercholesterolemia related gene variants: LDLR c.667G&amp;gt;A, PCSK9 c.658-36G&amp;gt;A, PCSK9 c.996+55C&amp;gt;A and APOB c.12382G&amp;gt;A between both cohorts. Conclusion A novel variant of SYNE1 gene is associated with myocardial infarction in young age patients with a family history of premature atherosclerosis.

Strategic and Innovative Roles of lncRNAs Regulated by Naturally-Derived Small Molecules in Cancer Therapy.

In the field of precision and personalized medicine, the next generation sequencing method has begun to take an active place as genome-wide screening applications in the diagnosis and treatment of diseases. Studies based on the determination of the therapeutic efficacy of personalized drug use in cancer treatment in the size of the transcriptome and its extension, lncRNA, have been increasing rapidly in recent years. Targeting and/or regulating noncoding RNAs (ncRNAs) consisting of long noncoding RNAs (lncRNAs) are promising strategies for cancer treatment. Within the scope of rapidly increasing studies in recent years, it has been shown that many natural agents obtained from biological organisms can potentially alter the expression of many lncRNAs associated with oncogenic functions. Natural agents include effective small molecules that provide anti-cancer effects and have been used as chemotherapy drugs or in combination with standard anti-cancer drugs used in routine treatment. In this review, it was aimed to provide detailed information about the potential of natural agents to regulate and/or target non-coding RNAs and their mechanisms of action to provide an approach for cancer therapy. The discovery of novel anti-cancer targets and subsequent development of effective drugs or combination strategies that are still needed for most cancers will be promising for cancer treatment.

Characterization of complete mitochondrial genome of three Horse flies of the genus Tabanus (Diptera: Tabanidae): comparative analysis.

Tabanidae (Horse-Flies or Deer-Flies) are one of the most economically important as well as medically significant haematophagous insect family within the order Diptera. Members of this group are also responsible for the mortality of substantial number of live-stock every year. Due to their pathogen transmission potential and vector competencies makes them an important insect group to study. Till now, mitochondrial genome of 18 species of tabanids were available. The complete mitogenome of three species T. diversifrons (15,809bp), T. rubidus (15,878bp) and T. tenens (15,872bp) were generated by Next generation sequencing method. They consist 37 genes, with a positive AT skew and a negative GC skew. The gene order of these three species is similar to the typical gene arrangement of infra-order Tabanomorpha. Most of the tRNAs showed typical clover-leaf secondary structure except trnS1, which lacks the DHU arm. The sliding window analysis showed that thenad4L is the most conserved while atp8, and nad6 are the most variable genes. Moreover, the ratios of non-synonymous to synonymous substitution rates indicated that all PCGs under the purifying selection. Phylogeny revealed Chrysops and Haematopota are monophyletic while species of Hybomitra are nested within the polyphyletic clade of Tabanus. T. diversifrons exhibits sister relationship with Atylotus miser. Two morphologically divergent species T. rubidus and T. tenens are found to be genetically similar and indistinguishable by mitochondrial genome. The hypervariable genes like atp8 and nad6 can be used as molecular markers for the identification of recently diverged lineages of family Tabanidae. Further, to address uncertainties arising from the two morphological divergent species, it is imperative to obtain data from nuclear gene markers.

Clinical case of combined genetic pathology in a child with jaundice syndrome

Diagnosis of a combined genetic pathology that occurs with jaundice syndrome is a difficult task, because of common clinical manifestations for several diseases having different genesis and requiring specific diagnosis and treatment tactics. Icteric staining of the skin due to hyperbilirubinemia at any age requires immediate examination to establish its genesis and differential diagnosis between infectious and non-infectious diseases. The spectrum of diseases accompanied by jaundice syndrome includes more than 50 different diseases. The main mechanisms of suprahepatic jaundice are: firstly, a violation of the capture and glucuronidation of bilirubin in the liver, and secondly, an increased destruction of red blood cells due to hemolysis or during the disposal of blood during internal bleeding. The first mechanism is realized in Gilbert’s syndrome, while the second mechanism of suprahepatic jaundice is often due to hemolytic anemia.In presented clinical case, the implementation of two pathogenetic forms of jaundice in one patient is shown — suprahepatic, caused by type 2 hereditary stomatocytosis and Gilbert’s syndrome, as well as obstructive jaundice, caused by cholelithiasis. The special attention should be paid to precise patient examination, if disease manifestations are not consistent with classical clinical picture. The use of a new generation sequencing method, along with traditional diagnostics methods, made it possible to establish an accurate diagnosis and determine the tactics of patient management.

Oral microbiota study of the patients after hospitalisation for COVID-19, considering selected dental indices and antibiotic therapy using the next generation sequencing method (NGS)

ABSTRACT Background Poor oral hygiene and the increased incidence and severity of periodontitis may exacerbate SARS-CoV-2 infection. The aim was to evaluate the oral microbiota of 60 participants divided into groups: COVID-19 convalescents who received antibiotics during hospitalization (I), COVID-19 convalescents without antibiotic therapy (II) and healthy individuals (III). Materials and Methods Dental examination was conducted, and oral health status was evaluated using selected dental indexes. Clinical samples (saliva, dorsal swabs, supragingival and subgingival plaque) were collected and used for metagenomic library to the next-generation sequencing (NGS) preparation. Results Each of the clinical materials in particular groups of patients showed a statistically significant and quantitatively different bacterial composition. Patients from group I showed significantly worse oral health, reflected by higher average values of dental indexes and also a higher percentage of Veillonella, Tannerella, Capnocytophaga and Selenomonas genera in comparison to other groups. Additionally, a statistically significant decrease in the amount of Akkermansia type in both groups with COVID-19 was observed for all materials. Conclusions The primary factor affecting the composition of oral microbiota was not the SARS-CoV-2 infection itself, but the use of antibiotic therapy. The increased percentage of pro-inflammatory pathogens observed in COVID-19 patients underscores the importance of preventing periodontal disease and improving oral hygiene in the future.