Generation Sequencing Methods Research Articles

Genetic profile of patients with stage I–IIIA non-small cell lung cancer

Background. The article presents data on somatic mutations found in the Russian population of patients with non-small cell lung cancer (NSCLC), their frequency, distribution depending on the histological type of tumor.Aim. To study the molecular genetic profile of Russian patients with stage I–IIIA localized NSCLC, the frequency of occurrence of various somatic mutations, variants of the switching status.Materials and methods. Genetic testing for a panel of 78 genes using the Next Generation Sequencing method of tumor material obtained after surgical treatment of 65 patients with localized stage I–IIIA NSCLC. Processing of the received data was carried out by methods of descriptive statistics.43Results. Mutations in the EGFR, ALK, ROS1, RET, BRAF, KRAS, MET, HER2 genes were found only in adenocarcinoma. Among non-targeted mutations in adenocarcinoma, TP53, STK11, FGFR3, EML4, NF1, RB1, and KMTC2 mutations were the most common. In squamous cell lung cancer, TP53, KMT2C, TSC1, EML4, PTEN, NF1, COL22A1, CDKN2A, RB1, BRCA1 were the most common. Mutations in the EGFR gene were most often associated with mutations in TP53 (30 % of cases), RB1 (15 % of cases), COL22A1 (15 % of cases); ALK was combined with TP53, NF1, WT1 – in 33 % of cases, ROS1 with DDR2 (33 % of cases), ERBB2 was combined with NTRK1, GPC3, HIP1, KIF5B, TP53, XPC, COL22A1 – in 14 % of cases, BRAF was most often associated with mutations in the TP53 gene (14 %) and COL22A1 (13.8 %); a mutation in the RET gene was associated with a TP53 mutation; ROS1 translocation in 50 % of cases was associated with mutations in the TRIM33 and TP53 genes.Conclusion. The data obtained give an idea of the frequency of occurrence of somatic mutations among Russian patients with NSCLC, which is important for the selection of diagnostic panels, interpretation of their results, and also potentially for the development of original custom Next Generation Sequencing testing panels.

Abstract 6086: PALB2 germline mutations in a large cohort Middle Eastern breast-ovarian cancer patients

Abstract Background: The PALB2 gene encoding protein is involved in the homologous recombination repair pathway and plays an important role in maintaining genomic integrity. PALB2 heterozygous pathological variants (PV) or likely pathogenic variants (LPV) are associated with increased risk of breast and ovarian cancer. However, the prevalence and clinicopathological association of PALB2 germline PV/LPV in Middle Eastern breast and ovarian cancers is not fully identified. Purpose: We retrospectively screened a cohort of 918 cancer patients (791 breast cancer and 127 ovarian cancer) from Saudi Arabia using targeted capture-based next generation sequencing (NGS) method for PALB2 germline variants regardless the family history of these patients or the presence of other hereditary cancer susceptibility genes mutations. Clinicopathological criteria were also fully analyzed. Results: Five heterozygous PVs or LPVs were identified in six cases, accounting for 0.65% (6/918) of entire cohort. Two cases (33.3%) harbored PVs and four cases (66.7%) carried LPVs. Four PVs/LPVs (80%) were frameshift along with one novel splicing LPV (c.2835-1 C>A). One recurrent LPV (c.3425delT) was identified in two cases. All six affected carriers have breast cancer diagnosis with median age of 39.5 years (range 34-49 years). Only two cases (33%) have documented family history of cancer. Breast cancer phenotype was invasive ductal unilateral cancer in all cases with 66.7% of hormone receptor positive and 16% of triple negative tumors. Conclusion: Germline PVs/LPVs in the PALB2 gene were observed in low frequency of 0.65% in Saudi breast and/or ovarian cancer. Our study confirms one recurrent LPV and one novel LPV in Saudi breast cancer patients. Citation Format: Rong Bu, Abdul K. Sira, Sandeep Parvathareddy, Kaleem Iqbal, Saud Azam, Zeeshan Qadri, Maha Al-Rasheed, Wael Haqawi, Mark Diaz, Ingrid G. Victoria, Khawla S. Al-Kuraya. PALB2 germline mutations in a large cohort Middle Eastern breast-ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6086.

Dynamics of oral microbiome acquisition in healthy infants: A pilot study.

The human oral microbiota is one of the most complex bacterial communities in the human body. However, how newborns initially acquire these bacteria remains largely unknown. In this study, we examined the dynamics of oral microbial communities in healthy infants and investigated the influence of the maternal oral microbiota on the acquisition of the infant's oral microbiota. We hypothesized that the infant oral microbial diversity increases with age. One hundred and sixteen whole-salivary samples were collected from 32 healthy infants and their biological mothers during postpartum and 9- and 15-month well-infant visits. Bacterial genomic DNA was extracted and sequenced by Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) methods. The Shannon index was used to measure the microbial diversity of the infant-mother dyads (alpha diversity). The microbial diversity between the mother-infant dyads (beta-diversity) was calculated using the weighted non-phylogenetic Bray-Curtis distance in QIIME 1.9.1. Core microbiome analysis was performed using MicrobiomeAnalyst software. Linear discriminant analysis coupled with effect size analysis was used to identify differentially abundant features between mother and infant dyads. A total of 6,870,571 16S rRNA reads were generated from paired mother-infant saliva samples. Overall, oral microbial profiles significantly differed between the mother and infant groups (p < 0.001). The diversity of the salivary microbiomes in the infants increased in an age-dependent manner, whereas the core microbiome of the mothers remained relatively stable during the study period. Breastfeeding and gender did not affect the microbial diversity in infants. Moreover, infants had a greater relative abundance of Firmicutes and a lower abundance of Actinobacteria, Bacteroidetes, Fusobacteria, and Proteobacteria than their mothers. The SparCC correlation analysis demonstrated constant changes in infants' oral microbial community network (p < 0.05). This study provides new evidence that the oral cavities of infants are colonized by a distinct group of bacterial species at birth. The acquisition and diversity of changes in oral microbial composition are dynamic during the first year of an infant's life. Before reaching the second birthday, the composition of the oral microbial community could be more similar to that of their biological mothers.

SUP: a probabilistic framework to propagate genome sequence uncertainty, with applications.

Genetic sequencing is subject to many different types of errors, but most analyses treat the resultant sequences as if they are known without error. Next generation sequencing methods rely on significantly larger numbers of reads than previous sequencing methods in exchange for a loss of accuracy in each individual read. Still, the coverage of such machines is imperfect and leaves uncertainty in many of the base calls. In this work, we demonstrate that the uncertainty in sequencing techniques will affect downstream analysis and propose a straightforward method to propagate the uncertainty. Our method (which we have dubbed Sequence Uncertainty Propagation, or SUP) uses a probabilistic matrix representation of individual sequences which incorporates base quality scores as a measure of uncertainty that naturally lead to resampling and replication as a framework for uncertainty propagation. With the matrix representation, resampling possible base calls according to quality scores provides a bootstrap- or prior distribution-like first step towards genetic analysis. Analyses based on these re-sampled sequences will include a more complete evaluation of the error involved in such analyses. We demonstrate our resampling method on SARS-CoV-2 data. The resampling procedures add a linear computational cost to the analyses, but the large impact on the variance in downstream estimates makes it clear that ignoring this uncertainty may lead to overly confident conclusions. We show that SARS-CoV-2 lineage designations via Pangolin are much less certain than the bootstrap support reported by Pangolin would imply and the clock rate estimates for SARS-CoV-2 are much more variable than reported.

Изучение структуры микробного сообщества черноземных почв Молдовы с использованием высокопроизводительного секвенирования

Based on the new generation sequencing method – pyrosequencing (Illumina), for the first time in Moldova, there were determined and described the metagenomic characteristics of the typical chernozem microbiome under different land use conditions. It was revealed that the prokaryotic community is represented by both domains, in particular, seventeen bacterial phyla (59.8%) and two phylum Archaea (6.2%), only nine phyla had an abundance above 1.0%: the archaeal phylum Thaumarchaeota and the bacterial phyla Proteobacteria, Actinobacteriota, Bacteroidota, Acidobacteriota, Firmicutes, Verrucomicrobiota, Planctomycetota, and Myxococcota. Phyla with a share of less than 1.0% preferred the conditions of experimental variants of crop rotations. A significant proportion were unclassified microorganisms, the abundance of which varied from 25.8 to 38.2% in spring and from 27.9 to 40.2% insummer. The conducted studies showed that the agrotechnical measures had a significant impact on the structure of the microbiome community of a typical chernozem.

Diagnostic Performance of Immunohistochemistry Compared to Molecular Techniques for Microsatellite Instability and p53 Mutation Detection in Endometrial Cancer.

Molecular algorithms may estimate the risk of recurrence and death for patients with endometrial cancer (EC) and may impact treatment decisions. To detect microsatellite instabilities (MSI) and p53 mutations, immunohistochemistry (IHC) and molecular techniques are used. To select the most appropriate method, and to have an accurate interpretation of their results, knowledge of the performance characteristics of these respective methods is essential. The objective of this study was to assess the diagnostic performance of IHC versus molecular techniques (gold standard). One hundred and thirty-two unselected EC patients were enrolled in this study. Agreement between the two diagnostic methods was assessed using Cohen's kappa coefficient. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the IHC were calculated. For MSI status, the sensitivity, specificity, PPV and NPV were 89.3%, 87.3%, 78.1% and 94.1%, respectively. Cohen's kappa coefficient was 0.74. For p53 status, the sensitivity, specificity, PPV, and NPV were 92.3%, 77.1%, 60.0% and 96.4%, respectively. Cohen's kappa coefficient was 0.59. For MSI status, IHC presented a substantial agreement with the polymerase chain reaction (PCR) approach. For the p53 status, the moderate agreement observed between IHC and next generation sequencing (NGS) methods implies that they cannot be used interchangeably.

Current Status and Prospects for Standards, Regulations, and Detection of Probiotic Yogurt: Review

Yogurt fermentation is known to be beneficial because it provides a low pH and harsh environment for foodborne pathogens and improves organoleptic properties. Additionally, organic acids produced through fermentation have a good effect on the viscosity and gelling properties of yogurt. Several potential health benefits of probiotic and generally recognized as safe strains have been suggested. Yogurt is the preferred vehicle for delivering probiotics to health-conscious consumers. Therefore, manufacturers of probiotic beverages must comply with the relevant regulations. The development of probiotic yogurt begins with the selection of strains with safety and functional properties of probiotics. The selected probiotic strain should be technically suitable for viability and improve organoleptic quality while maintaining the number of bacteria above the standard value during processing and storage conditions. In addition, the efficacy of probiotic strains contained in yogurt should be investigated, confirmed, and approved according to well-designed clinical trials. Although various methods are used to detect probiotic strains, the recently widely used next generation sequencing method can be actively utilized. In the future, more research should be conducted with the latest methods to identify probiotic functions and accurately detect probiotic strains.

Importance of the female reproductive tract microbiome and its relationship with the uterine environment for health and productivity in cattle: A review

Once thought to be sterile, the reproductive tract microbiome has been characterized due to the transition from culture-dependent identification of bacteria to culture-independent sequencing methods. The urogenital microbiome was first identified in women through the Human Microbiome Project, which led to research in other species such as the bovine. Previous research focused on uterine bacteria associated with postpartum disease, but next generation sequencing methods identified a normal, healthy bacterial community of the reproductive tract of cows and heifers. Bacterial communities are now understood to differ between the uterus and vagina, and throughout the estrous cycle with changes in hormone dominance. In a healthy state, the bacterial communities largely interact with the uterine environment by assisting in maintaining the proper pH, providing and utilizing nutrients and metabolites, and influencing the immunological responses of the reproductive tract. If the bacterial communities become unbalanced due to an increase in potentially pathogenic bacteria, the health and fertility of the host may be affected. Although the presence of a reproductive tract microbiome has become widely accepted, the existence of a placental microbiome and in utero colonization of the fetus is still a popular debate due to conflicting study results. Currently, researchers are evaluating methods to manipulate the reproductive bacterial communities, such as diet changes and utilizing probiotics, to improve reproductive outcomes. The following review discusses the current understanding of the reproductive tract microbiome, how it differs between humans and cattle, and its relationship with the uterine environment.

High throughput single-molecule technology.

Single-molecule fluorescence resonance energy transfer (smFRET) has been an indispensable tool to probe the structure of biomolecules on the nanometer scale. The intrinsic low-throughput nature of single molecule observation has, however, prevented the technique to be used to study a large pool of samples. Here, we present a highly parallel smFRET measurement scheme by combining the conventional smFRET measurement with a next generation sequencing method. A library of DNA molecules, each of which carries one of the thousands of different sequences of interest, was prepared and measured in a one pot recipe. By using this high-throughput approach, we experimentally determine the sequence-dependent end-to-end distance of single stranded DNA in various native aqueous buffer conditions.

Molecular genetic sequencing of neuroendocrine tumors of the gastrointestinal tract.

651 Background: The objective of this work was to evaluate the molecular and genetic features of the NET gastrointestinal tract. Genomic sequencing of 30 tumor samples of the latest generation was carried out and the data obtained were evaluated. Methods: In total, 30 tumor blocks of patients with gastrointestinal NET were tested by the new generation sequencing (NGS) method. The following genes were analyzed: ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CHEK1, CHEK2, EPCAM, FANCL, MLH1, MSH2, NBN, NF1, PALB2, PMS2, RAD51B, RAD51C, RAD51D, RAD54L, STK11, TP53, POLE, KRAS, NRAS, BRAF, EGFR, ERBB2, PIK3CA, MET ex14, BAT25, BAT26, NR21, NR24, MONO27, KIT, PDGFRA, Pi3Ca. Preparation of libraries for sequencing was carried out using NimblGen SepCapEZ Choice. Sequencing was performed on the Illumina MiSeq device. Results: In the study cohort of patients whose tumor blocks were sent for examination by the NGS method, there were more women – 19 (63.3%) than men – 11 (36.7%). When analyzing the age of patients, it should be noted that among all age groups, elderly patients predominated (60-74 years according to the WHO classification, 2016) – 12 (40%). In the majority of patients – 10 (33.3%) – the primary tumor focus was localized in the pancreas, in 6 (20%) patients it was primarily affected stomach, 7 (23.3%) – small intestine. Other parts of the gastrointestinal tract were affected much less frequently: the colon – in 1 (3.3%) patients. According to the division according to the degree of differentiation of gastrointestinal NET10 tumors had a degree of differentiation G1 (33.3%), 16 -G3 (54.3%), 4– G3 (13.3%). In the tumor sample of a patient with gastric NET G1 (Ki 67 = 2%), POLE mutations were found: c.2276G &gt; A, p.R759H and CHEK2: c.470T &gt; C, p.I157T. Also, mutations MLH1: c.199G &gt; A, p.G67R BRCA1: c.1881 were found in the tumor sample of another patient with gastric NET G2 (Ki 67 = 7%). Conclusions: To improve the survival rates of patients, it is worth paying special attention to the molecular genetic study of the NET gastrointestinal tract. It is molecular genetic profiling that can become the basis for building more individualized algorithms for treating patients. The results of this study allow us to count on the fact that when conducting a study on a larger cohort of patients with gastrointestinal NET, it is possible to obtain results that will expand our knowledge of this nosology and allow us to supplement the already available information about prognosis factors and predictors of response to treatment.

Determination of Genotypes in Cytomegalovirus (CMV) Strains Obtained from Pediatric and Adult Immunocompromised Patients

Background and Purpose: Cytomegalovirus causes asymptomatic disease in individuals with normal immune system; and leads to serious complications in immunocompromised individuals and fetus. In CMV, gB is the most studied glycoprotein in terms of genotyping. Up to now, four different gB genotypes (gB 1-4) of CMV have been identified. &#x0D; In this study, it was aimed to determine the genotypes of CMV strains obtained from patients with immune deficiency.&#x0D; Methods: Twenty children and 29 adults, 49 patients who were followed in the Department of Adult Hematology and Pediatric Hematology were included in the study. DNA isolation was performed from samples with CMV DNA level of 1000 IU / ml and above, and 474 bp region from the gB region of the virus was amplified by nested PCR. This region was sequenced by the Sanger (ABI 3500 Prism) sequencing. Next generation sequencing (NGS) method was applied to the samples that CMV genotype could not be determined by Sanger sequencing.&#x0D; Results: Distribution of CMV genotypes of patients determined by Sanger sequencing; while it was determined as 18/49 (36.7%) type 1, 5/49 (10.2%) type 2, 5/49 (10.2%) type 3 and 1/49 (2%) type 4; 14/49 (28.5%) of them were found as mixed genotypes. CMV genotype could not be determined in 6 patients by Sanger sequencing and CMV genotype of these 6 patients were found as mixed genoype by NGS. Mixed genotype was detected in 20 (40.9%) of 49 patients, in total by Sanger sequencing and NGS. &#x0D; Conclusion: It is remarkable that the most common genotype is mixed genotype. It was concluded that it is appropriate to study the next generation sequencing method in patients whose CMV genotype cannot be determined by Sanger sequencing.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

FastContext: A tool for identification of adapters and other sequence patterns in next generation sequencing (NGS) data.

The development of next generation sequencing (NGS) methods has created the need for detailed analysis and control of each protocol step. NGS library preparation protocols may include steps with incorporation of various service sequences, such as sequencing adapters, primers, sample-, cell-, and molecule-specific barcodes. Despite a fairly high level of current knowledge, during the protocol development process researches often have to deal with various kinds of unexpected experiment outcomes, which result either from lack of information, lack of knowledge, or defects in reagent manufacturing. Detection and analysis of service sequences, their distribution and linkage may provide important information for protocol optimization. Here we introduce FastContext, a tool designed to analyze NGS read structure, based on sequence features found in reads, and their relative position in the read. The algorithm is able to create human readable read structures with user-specified patterns, to calculate counts and percentage of every read structure. Despite the simplicity of the algorithm, FastContext may be useful in read structure analysis and, as a result, can help better understand molecular processes that take place at different stages of NGS library preparation. The project is open-source software, distributed under GNU GPL v3, entirely written in the programming language Python, and based on well-maintained packages and commonly used data formats. Thus, it is cross-platform, may be patched or upgraded by the user if necessary. The FastContext package is available at the Python Package Index (https://pypi.org/project/FastContext), the source code is available at GitHub (https://github.com/regnveig/FastContext).

Effect of galangal essential oils on rumen microbial population and biodiversity on in vitro rumen fermentation

The study aimed to evaluate the effect of administering galangal essential oil (EO) on the abundance of rumen bacteria using the 16s rRNA method. The treatments included a control (no EO addition), galangal EO (30, 60, 120 µL), and cineole (5 µL). The treatments were assessed using a 48‐hour in vitro batch culture of rumen fluid containing a 60:40 ratio of forage to concentrate. For amplification of the prokaryotes (bacteria and archaea) in region V4, 16s rRNA primer 5’GTGCCAGCMGCCGCGTAA, GGACTACHVGGGTWTCTAAT3’ was employed. The data for rumen microbial abundance were analysed descriptively, while the data for rumen microbial diversity were obtained from the report on the Next Generation Sequencing Method. The microbial composition of each sample was tested for operational taxonomic units (OTUs) with a 97% identity rate on a valid label. The 16S rRNA gene sequencing yielded a total of 3,977 OTUs. Adding galangal and cineole EOs resulted in the same variation of the Shannon index. The population index (chao1 index) was highest when 60 µL of galangal EO was added, compared to 30 and 120 µL of galangal EO and cineole. In addition, providing 60 µL of galangal EO decreased the abundance of Prevotella ruminicola compared to the control and cineole doses. The addition of galangal EO also led to a decline in the number of Methanobacteriales. The population of the fibre‐degrading bacteria group (Ruminococcus albus and Ruminococcus flavefaciens) was higher in a dose of galangal EO than the control and cineole. Therefore, it can be concluded that the effective dose of galangal EO, i.e. 60 µL/300 mg (DM feed) in vitro, can reduce the abundance of Prevotella bacteria and methanogens.

Assessing the hidden diversity underlying consensus sequences of SARS-CoV-2 using VICOS, a novel bioinformatic pipeline for identification of mixed viral populations.

IntroductionCoinfection with two SARS-CoV-2 viruses is still a very understudied phenomenon. Although next generation sequencing methods are very sensitive to detect heterogeneous viral populations in a sample, there is no standardized method for their characterization, so their clinical and epidemiological importance is unknown. Material and methodsWe developed VICOS (Viral COinfection Surveillance), a new bioinformatic algorithm for variant calling, filtering and statistical analysis to identify samples suspected of being mixed SARS-CoV-2 populations from a large dataset in the framework of a community genomic surveillance. VICOS was used to detect SARS-CoV-2 coinfections in a dataset of 1,097 complete genomes collected between March 2020 and August 2021 in Argentina. ResultsWe detected 23 cases (2%) of SARS-CoV-2 coinfections. Detailed study of VICOS's results together with additional phylogenetic analysis revealed 3 cases of coinfections by two viruses of the same lineage, 2 cases by viruses of different genetic lineages, 13 were compatible with both coinfection and intra-host evolution, and 5 cases were likely a product of laboratory contamination. DiscussionIntra-sample viral diversity provides important information to understand the transmission dynamics of SARS-CoV-2. Advanced bioinformatics tools, such as VICOS, are a necessary resource to help unveil the hidden diversity of SARS-CoV-2.

A decade of gene discovery by the Alzheimer’s Disease Sequencing Project

AbstractBackgroundAlzheimer’s disease (AD) is caused in part by variation in genes and genetic regulatory elements. Early work identified autosomal dominant mutations in APP, PSEN1 and PSEN2 that cause AD, and large effect size variants in APOE. However, most of AD genetic risk is caused by variants that result in a modest change in risk. These variants are spread across many genetic elements. In 2009, genome‐wide association studies (GWAS) began to identify these AD loci (&gt; 60 sites). While these studies continue to be productive, GWAS lacks the resolution to identify the critical AD genes and causative variants responsible for risk/protective signals. In 2012, the Alzheimer’s Disease Sequencing Project (ADSP) was initiated to use Next Generation Sequencing methods for AD gene discovery. Sequence data has single nucleotide resolution with the potential to identify causative common and rare variants and to detect structural variants (SVs; insertions, deletions, copy number variation, etc.).MethodThe ADSP is a collaborative network of investigators that assembles cohorts for sequencing, processes the data, and performs genetic analysis. In the past 10 years, the ADSP generated whole exome (WES) and whole‐genome‐sequence (WGS) data for 20,504 case and 43,004 control subjects. Subjects sequenced are or clinically diagnosed or neuropathologically confirmed AD cases and cognitively normal elderly subjects. Subjects include non‐Hispanic whites, Hispanics, African Americans, and Asians. In progress are WGS data for an additional ∼20,000 subjects. Sequence data are harmonized by the Genome Center for Alzheimer Disease and shared with the research community via the National Institute on Aging Genetics Data Storage site.ResultAnalysis of WES and WGS data nominates 46 AD genes and regions of interest (PMID: 35005195). These genes implicate known and novel AD pathways providing new insight into AD pathogenesis and novel therapeutic targets. SVs were detected in ABCA7 and NCK2. The ADSP includes functional genomics studies to connect risk variants to specific genes and to understand the function of these variants. Analysis methods also includes artificial intelligence/machine learning approaches.ConclusionThe genes and pathways identified by the ADSP are a rich source of validated targets for future therapeutic development.

Co-Mutation Landscape and Clinical Significance of RAS Pathway Related Gene Mutations in Patients with Myelodysplastic Syndrome

Background Single gene mutations in the RAS pathway are uncommon and of unknown significance in myelodysplastic syndrome (MDS) patients, RAS pathway-related gene mutations (RASwaymut) as a whole may be significant and require further elucidation. Objective To explore the clinical characteristics and co-mutation landscape of MDS patients in the background of RASwaymut. Methods The clinical data of 370 MDS patients who were newly diagnosed between November 1, 2016 and August 31, 2020 in our hospital were collected and retrospectively reviewed. Next generation sequencing (NGS) method was used to detect a panel of 96 selected MDS-related gene mutations. Results RASwaymut was detected in 15.41% (57/370) of primary MDS patients. Male dominance (78.95%.vs 64.54%, P=0.03), higher median percentage of marrow blasts (2% vs 1%, P=0.00) and more higher risk patients according to revised international scoring system(IPSS-R) (80.70% .vs 59.11%, P=0.002) as well as higher acute myeloid leukemia (AML) transformation rate (35.09% .vs 14.38%, P=0.02) were observed in patients with RASwaymut when compared to those with wild type RAS pathway-related genes (RASwaywt). The most common co-mutated genes were ASXL1 (28.6%); TET2 (23.2%); U2AF1, RUNX1, TP53 (14.3%); DNMT3A (12.5%); ZRSR2 (10.7%); among which ASXL1 mutation rate were significantly higher than those with RASwaywt (P＜0.05). RASwaymut had no significant effect on response to disease-modifying regimens in MDS patients. However, Overall survivals (OS) of RASwaymut patients were significantly shorter than those with RASwaywt (16.05m .vs 92.3m, P=0.00), mainly in patients with blast cells less than 5% (P=0.002), normal karyotype (P=0.01) and lower IPSS-R risk (P=0.00). While multivariate prognostic analysis showed that RASwaymut lost its effect on OS, and just when it co-mutated with TET2 was an independent poor prognostic factor for all MDS patients (P=0.00, HR=4.77 with 95%CI: 2.4-9.51) and RASwaymut patients (P=0.02, HR 2.76, 95%CI 1.21-6.29). Conclusion RASwaymutwas associated with higher marrow blast cells and IPSS-R risk, higher incidence of leukemic transformation thus shorter OS in MDS patients, it could be viewed as a whole to predict poor prognosis. Co-mutation with TET2 may promote disease progression and was an independent poor prognostic factor in MDS patients.

English

A variant is an alternative nucleotide located at a specific region of a gene. 48 genes encode for human red cell blood group systems. Variants within these genes encode for alleles, which can be highly polymorphic. The blood group gene loci jointly display all types of inherited variants to include single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants. In Africa, there is limited information on the red cell variants. The aim of the study was to determine the incidence of Kell red blood cell variants among the Kenyans. Blood donor&rsquo;s samples that were used for routine grouping process were identified for this study. Next generation sequencing method was employed to predict the Kell red cell variants in blood donor samples. Descriptive statistics was applied and the result was presented in form of tables. The study reveals that Kell system has six variants with two major phenotypes and genotpes. The most common phenotype genotyped was KEL2 (KEL*02/KEL*02) 79% (85/108) followed by KEL2 KEL6 KEL7 (KEL*02.06/KEL*02) 16.7% (18/108). The rest were found to be of low frequency and all were associated with KEL2. The study recommends an extended study with a large sample size and introduction of extended phenotyping to aid Kell antigens identification in the donor population. &nbsp; Key words: Kell, variant, allele, KEL, KEL*02/KEL*02, molecular, genome, gene, next generation sequencing.

Allele and eight-locus haplotype frequencies in population of Belgorod region, Russia

This paper reports the HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and –DPB1 alleles and estimated haplotype frequencies in a population of 153 healthy potential blood marrow donors from Belgorod region, Russia. HLA genotyping was performed by next generation sequencing method (NGS). Statistical analysis were performed using Arlequin software packages. There was no deviation from Hardy-Weinberg Equilibrium detected at HLA-A, -B, -DRB1, -DQA1, -DQB1, and -DPA1 loci. Deviation was detected at the HLA-C and DPB1 locus probably due to an excess of C*12:03:01 and DPB1*04:01:01 homozygotes. These genotype data are available in the Allele Frequencies Net Database under the population name, “Russia, Belgorod Region” and the identifier: 3780.

The origin of the Qajar Shah dynasty according to their Y-DNA

The subject of the study was the genealogy of the Qajar dynasty, which ruled Iran in 1795-1925. Documentary sources indicate Gara Piri bey Qajar (XV century – 1513), the first beglyarbek of Karabakh with the center in Ganja (now Azerbaijan) as the earliest ancestor of the dynasty. At the end of the XVI century, the great–grandson of Gar Piri bey was appointed Shah Abbas I beglyarbek of Astrabad (now Gorgan, Iran), from whose descendant - Agha Muhammad Khan Qajar (1741-1797), the shah dynasty began. Agha-Mohammed Khan himself traced his family back to a legendary ancestor named Qajar, the son of Sartak-noyon from the Mongolian Jalai tribe, the mentor of Genghisid Argun Khan (1284-1291). According to other sources, the Qajars are a Turkoman tribe that settled in Transcaucasia during the Mongol period. These data on the early history point to the Qajar dynasty as having originally Central Asian origin. In 2007, a study of the Y-DNA of two modern representatives of different lines of the Qajar dynasty was conducted. Tests have shown that both lines really originate from a recent common paternal ancestor and belong to haplogroup J1-M267, widespread in the Middle East. However, apart from the genetic confirmation of the common paternal origin of these two lines and the declaration of the marginality of the ancestral legend, there is virtually no analysis in the work that gives a definitive answer to the question of the origin of the dynasty itself. In the development of this work, a more in-depth study of Y-DNA by new generation sequencing methods was carried out. The paternal origin of the Qajar dynasty from the indigenous population of the northern regions of Azerbaijan has been revealed.