Generation Sequencing Methods Research Articles

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 <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 <65 years in women or <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>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>A, PCSK9 c.658-36G>A, PCSK9 c.996+55C>A and APOB c.12382G>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.

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.

Preliminary Information of Iranian Lizard Leishmania Promastigote Transcriptome Sequencing by Next Generation Sequencing (NGS) Method.

A lizard Leishmania has been isolated from a lizard (Agama agilis) in Iran. Its genome sequence has not been determined, so far. The study was done at Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2017-2023. Leishmania promastigotes were cultured in RPMI1640 culture medium and collected at logarithmic phase by centrigugation. Parasite RNA was extracted by the Qiagene standard kit and its quantity and quality was determined and sequenced by NGS method with Illumina PE machine at BGI Company (China). The number of 8316 mRNA, 83 tRNA, 63 rRNA, 83 ncRNA, 5 snRNA, 1039 snoRNA, 36 region, and 3 repeat regions, 8343 CDS, 9597 Exon and 9292 Genes were identified in promastigote of Iranian lizard Leishmania. Genomic elements of Iranian lizards Leishmania (with unique characteristics) were determined and identified by NGS system.

Інноваційна діагностика розладів нейророзвитку в дітей

Today, genetic diagnosis methods are a powerful tool for a practicing doctor, which allows not only to establish the etiology of neurodevelopmental disorders in children, but also influences the further tactics of patient management, the choice of further diagnostic and therapeutic interventions, helps in predicting subsequent pregnancies for the family. Purpose - to analyze the information of modern specialized literature regarding the features of the use of modern cytogenetic methods of diagnosis, in particular, chromosomal micrometric analysis (СMA); give a clinical example of a child with a neurodevelopmental disorder and established chromosomal etiology. A review of the scientific literature regarding the advantages and features of using innovative methods of cytogenetic diagnostics (karyotyping, СMA), clinical manifestations of 2q13 microdeletion syndrome is given. A description of the clinical case of diagnosis of a child with 2q13 microdeletion syndrome is presented. A chromosomal micrometric analysis was performed, which revealed a heterozygous microdeletion on 2q13 with a size of 122 kilobases, which led to the loss of the NPHP1 and MALL genes. The obtained result of the CMA made it possible to optimize the tactics of monitoring the child, taking into account the increased risk of the development of kidney pathology and leukoencephalopathy, the recommended annual determination of the level of creatinine and urea in the blood, conducting an ultrasound of the kidneys and an MRI of the brain. Conclusions. The given clinical observation confirms the complexity of the diagnostic search for neurodevelopmental disorders in children. In children with autism spectrum disorder, developmental delay, intellectual disability, in the absence of epileptic seizures, regardless of the presence of dysmorphic facial features, it is recommended to begin the examination with CMA, and in children with epileptic encephalopathies, it is optimal to begin the examination with the next generation sequencing method (NGS), namely whole exome sequencing. The research was carried out in accordance with the principles of the Declaration of Helsinki. Informed consent of the children’s parents was obtained for the research. No conflict of interests was declared by the authors.

Diversity of Bacteroidaceae family in gut microbiota of patients with chronic kidney disease and end stage renal disease.

Human intestine microbiota are known to be directly and indirectly altered during some diseases such as kidney complications. Bacteroides is considered as the main and the most abundant phylum among human gut microbiota, which has been classified as enterotype 1. This study aimed to assess the abundance of Bacteroides spp. in fecal flora of end-stage renal disease (ESRD) and chronic kidney disease (CKD) patients and compare it with the Bacteroides composition among fecal flora of healthy individual. Fresh fecal samples were collected from 20 CKD/ESRD patients and 20 healthy individual without any kidney complications. The pure microbial DNA was extracted by QIAamp Stool Mini Kit from stool samples. MiSeq system was used to analyze the intestinal composition by next generation sequencing method. A number of 651 bacterial strains were isolated and identified from 40 fecal samples of both patients and healthy groups. Bioinformatics analysis defined 18 different types of Bacteroides species which included 2.76% of all strains. Statistical analysis showed no significant difference between study groups (P>0.05). In both healthy and patient groups three species including B. dorei, B. uniformis, and B. ovatus have allocated the most abundance to themselves. The lowest abundance was related to B. eggerthii, A. furcosa and B. barnesiae among CKD/ESRD patients and A. furcosa, B. barnesiae, and B. coprocola had the lowest abundance among healthy people. This study indicates despite all previous evidence of Bacteroides role in gut microbiota, it had no different distribution between healthy persons and CKD/ESRD patients.

Characterization of Mutation of BRCA1 Gene at Chromosome 17q21 in Bangladeshi Women With Familial Breast Cancer

Breast cancer is the most common cancer among women in Bangladesh. About 69% cancer death of women is due to breast cancer. In Bangladesh the incidence rate of breast cancer is about 22.5 per 100000 female. Female who carries mutation in the BRCA1 gene have an 80% chance of developing breast cancer during their lifetime. Due to its high prevalence, genetic testing for BRCA1 mutation has become an integral part of clinical practice. This study was aimed to characterize the mutation at chromosome 17q21 by targeting BRCA1 gene among Bangladeshi women with familial breast cancer. This descriptive case series study was conducted in the Department of Biochemistry, Dhaka Medical College, Dhaka, from July 2017 to June 2018. In this study, thirty diagnosed breast cancer patients with family history breast cancer were selected according to the selection criteria from the Department of Surgery and Department of Radiotherapy, Dhaka Medical College, Dhaka. Mutations were detected by Next Generation Sequencing method. Characterization and clinical significance were verified from NCBI genome database. The study parameters were presence of mutation, types of mutation, clinical significance, frequency (%) of mutation. In this study, sixteen distinct mutations were found. Three pathogenic mutations (10%) were found among the patients which were nonsense mutations. Pathogenic mutations were c.5251C>T, c.2158G>T and c.1059G>A. Three mutations of uncertain significance with missense variant, five benign mutations of missense variant and five benign mutations of silent variant were also found among thirty patients. In this study we found that, Bangladeshi women with familial breast cancer had 10% pathogenic mutations. Early screening for BRCA1 gene mutations might provide better information for health to those who have family history of breast cancer.
 Bangladesh J Med Biochem 2018; 11(2): 17-23

The effect of various additives on the microbiota of ruminant rumen and the course of metabolic processes in the rumen <i>in vitro</i> studies

Relevance. In an in vitro study, changes in the taxonomic scar and the course of fermentation processes in response to the addition of various lipid additives to the ruminant diet were studied.Methods. Three samples were formed: control — without the addition of oil, the experimental ones were supplemented with sunflower oil (3%) (I) and soy (3%) (II). The object of the study is the scar content obtained from Kazakh white-headed bulls with chronic scar fistula (n = 3) by the Latin square method. Studies of the digestibility of SV were carried out using the incubator «ANKOM DaisyII» according to a specialized technique. Laboratory studies: the level of volatile fatty acids (VFA) in the contents of the scar was determined by gas chromatography on a gas chromatograph «Crystallux-4000M», determination of nitrogen forms according — to GOST 26180-84. Microbial biodiversity of the rumen contents was carried out using MiSeq («Illumina», USA) by the new generation sequencing method (NGS) with a set of reagents MiSeq® Reagent Kit v3 (600 cycle).Results. The addition of oils had a stimulating effect on the digestibility of SV feed in the rumen, increasing this indicator by 2.7%. Compared with the control, the addition of oil reduced the concentration of total and protein nitrogen in the scar content, while the content of non-protein nitrogen increased (by 5.4%) when using sunflower and (by 7.8%) soybean oil. The additional inclusion of oils showed an increase in the level of acetic and butyric acids, respectively: in the group using sunflower oil — by 46.8% and 55.6%, in the group with soybean oil — by 51.5% and 60.5% relative to the control. The additional inclusion of sunflower oil in the diet showed a change in the number of microorganisms in the RYE, but the qualitative composition of the microbiota relative to the control group did not change significantly.

Whole Exome Sequencing to Find Candidate Variants for the Prediction of Kidney Transplantation Efficacy.

Kidney transplantation is the optimal treatment strategy for some end-stage renal disease (ESRD); however, graft survival and the success of the transplantation depend on several elements, including the genetics of recipients. In this study, we evaluated exon loci variants based on a high-resolution Next Generation Sequencing (NGS) method. We evaluated whole-exome sequencing (WES) of transplanted kidney recipients in a prospective study. The study involved a total of 10 patients (5 without a history of rejection and 5 with). About five milliliters of blood were collected for DNA extraction, followed by whole-exome sequencing based on molecular inversion probes (MIPs). Sequencing and variant filtering identified nine pathogenic variants in rejecting patients (low survival). Interestingly, in five patients with successful kidney transplantation, we found 86 SNPs in 63 genes 61 were variants of uncertain significance (VUS), 5 were likely pathogenic, and five were likely benign/benign. The only overlap between rejecting and non-rejecting patients was SNPs rs529922492 in rejecting and rs773542127 in non-rejecting patients' MUC4 gene. Nine variants of rs779232502, rs3831942, rs564955632, rs529922492, rs762675930, rs569593251, rs192347509, rs548514380, and rs72648913 have roles in short graft survival.

Comparing the melissopalynological and next generation sequencing (NGS) methods for the determining of botanical origin of honey

Comparing the melissopalynological and next generation sequencing (NGS) methods for the determining of botanical origin of honey

A universal lateral flow assay for microRNA visual detection in urine samples

MicroRNAs (miRNAs) have been emerged as novel and significant biomarkers in liquid biopsy that can be found in different body fluids. Several techniques have been developed and applied for miRNAs analysis, including nucleic acid-based amplification methods, next generation sequencing, DNA microarrays and new genome-editing methods. These methods, however, are time-consuming and require expensive instruments and specially trained personnel. Biosensors, on the other hand, are alternative and valuable analytical/diagnostic tools due to their simplicity, cost-effectiveness, rapid analysis and ease of use. Several biosensors, especially nanotechnology-based ones, have been developed for miRNA analysis that are based either on target amplification or signal amplification and target re-cycling for sensitive detection. At this point of view, we have introduced a new and universal lateral flow assay in combination with reverse transcription – polymerase chain reaction (RT-PCR) and gold nanoparticles as reporters for the detection of miR-21 and miR-let-7a in human urine. It is the first time that such a biosensor has been applied to the detection of microRNAs in urine. As low as 102-103 copies of miR-21 and 102--104 copies of miR-let-7a added in urine were detectable by the proposed lateral flow assay with great specificity and repeatability (%CVs <4.5%).

Perspectives on Sampling and New Generation Sequencing Methods for Low-Biomass Bioaerosols in Atmospheric Environments.

The online version contains supplementary material available at 10.1007/s41745-023-00380-x.

UORF4u: a tool for annotation of conserved upstream open reading frames.

Upstream open reading frames (uORFs, often encoding so-called leader peptides) can regulate translation and transcription of downstream main ORFs (mORFs) in prokaryotes and eukaryotes. However, annotation of novel functional uORFs is challenging due their short size of usually less than 100 codons. While transcription- and translation-level next generation sequencing (NGS) methods can be used for genome-wide functional uORF identification, this data is not available for the vast majority of species with sequenced genomes. At the same time, the exponentially increasing amount of genome assemblies gives us the opportunity to take advantage of evolutionary conservation in our predictions of functional ORFs.Here we present a tool for conserved uORF annotation in 5' upstream sequences of a user-defined protein of interest or a set of protein homologues. It can also be used to find small conserved ORFs within a set of nucleotide sequences. The output includes publication-quality figures with multiple sequence alignments, sequence logos and locus annotation of the predicted conserved uORFs in graphical vector format. uORF4u is written in Python3 and runs on Linux and MacOS. The command-line interface covers most practical use cases, while the provided Python API allows usage within a Python program and additional customisation. Source code is available from the GitHub page: github.com/GCA-VH-lab/uorf4u Detailed documentation that includes an example-driven guide available at the software home page: gca-vh-lab.github.io/uorf4u. A web version of uORF4u is available at server.atkinson-lab.com/uorf4u.

Method of DNA Extraction from Plant for Metagenomic Analysis as Example from Grape &lt;i&gt;Vitis amurensis&lt;/i&gt; Rupr.

A new method for extracting DNA from plants is proposed, using the example of wild grapes Vitis amurensis Rupr., for further preparation of libraries for metagenomic analysis. The method is based on the isolation of DNA by an inexpensive CTAB method with an additional stage of DNA purification using silica spin columns (CTAB spin method). A comparative analysis of the results of metagenomic analysis of endophytes on DNA isolated using the proposed CTAB-spin method and using the commercial set ZymoBIOMICS DNA Miniprep (Zymo Research). It was found that when using the CTAB-spin method, the number of sequences of the 16S rRNA site and the diversity of bacterial genera were 2.8 and 1.2 times greater, respectively, than when using the ZymoBIOMICS kit. At the same time, the number of sequences of the internal transcribed spacer 1 (ITS1) and the biodiversity of endophytic fungi did not differ significantly during DNA extraction by two methods. Thus, the proposed method of DNA isolation for metagenomic analysis is an available and effective alternative to commercial kits for the isolation of plant DNA for new generation sequencing methods.