Genome Sequencing Techniques Research Articles

Revisiting the hallmarks of cancer: A new look at long noncoding RNAs in breast cancer.

Breast cancer is one of the leading causes of death in women worldwide. The increasing understanding of the molecular mechanisms underlying its heterogeneity favors a better understanding of tumor biology and consequently the development of better diagnostic and treatment techniques. The advent of tumor genome sequencing techniques has highlighted more participants in the process, in addition to protein-coding genes. Thus, it is now known that long noncoding RNAs, previously described as transcriptional noise with no biological function, are intimately associated with tumor development. In breast cancer, they are abnormally expressed and closely associated with tumor progression, which makes them attractive diagnostic biomarkers and prognostic and specific therapeutic targets. Therefore, a thorough understanding of the regulatory mechanisms of long noncoding RNAs in breast cancer is essential for the search for new treatment strategies. In this review, we summarize the major long noncoding RNAs and their association with the cancer characteristics of the ability to sustain proliferative signaling, evasion of growth suppressors, replicative immortality, activation of invasion and metastasis, induction of angiogenesis, resistance to cell death, reprogramming of energy metabolism, genomic instability and sustained mutations, promotion of tumor inflammation, and evasion of the immune system. In addition, we report and suggest how they can be used as prognostic biomarkers and possible therapeutic targets.

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.

Espécies fitopatogênicas de Phytophthora

The genus Phytophthora accommodates six of the ten species of oomycetes listed as the most important for their destructive potential to agriculture. Phytopathogens of this genus attack a wide range of hosts, where they cause seed and root rot, seedling damping off, collar, tuber and rhizome rot and soft rot in fruits in contact with the soil. Although they are basically considered soil pathogens, some species can attack the aerial part of their hosts and cause leaf blight and fruit, stem and flower rot. The management of diseases caused by Phytophthora spp. can be difficult because it is an inhabitant of the soil where it survives for long periods through resistance structures and because of its polyphagous nature. Since the first description of the genus, almost a century and a half ago, this oomycete has been widely studied, with significant advances in the understanding of biology, physiology and genetic variability. More recently, with the use of genomic sequencing techniques, it has been possible to establish the phylogenetic relationships of Phytophthora spp. with other organisms at different taxonomic levels. These studies continue to expand widely, as organisms are constantly exposed to evolutionary processes. In this sense, with the advances in the use of molecular markers, it has been possible to identify new species, phytopathogens and inhabitants of aquatic and forest environments, and also to resolve some species complexes. This review aimed to compile the state of the art of the advances obtained in the studies of Phytophthora spp., focusing on research collaboration in understanding the pathosystems involving this genus in the main species of cultivated plants

Changing trends in the diagnosis of genitourinary tuberculosis in post Covid-19 pandemic era

efforts to achieve a TB-free world by 2030. The health professionals and the health care support team suffered a major setback in reaching out to the patients due to various restrictions imposed on them during this pandemic period. Patients found it difficult to reach out to the health care team, owing largely to the fear factor and also due to inaccessibility to health care services. The delay in TB notifications, emergence of drug resistant strains and restrictions in the supply- chain added further to the difficulties faced. India is a nation with one of the largest numbers of TB victims. The strategic measures taken by the Government of India (GOI) took a sudden jolt with the onset of the COVID-19 pandemic. The TB elimination measures reverted back to TB containment measures. Various African countries including Ethiopia recorded a massive fall out of more than 75% during the various lockdown measures imposed. However, India took collateral measures to combat this setback. Its National Tuberculosis Elimination Programme (NTEP) involved private players to combat TB epidemic, recruited Patient provider support agencies and actively engaged Private Sectors through innovative patient provider incentives to bring down numbers of the TB victims. The diagnostic methods that took about 4 to 6 weeks to confirm the diagnosis of TB had earlier put the diagnosis and management of TB to the back seat. However, recent innovations in the molecular biology, immunology and genomic sequencing techniques have greatly aided an earlier and accurate diagnosis of tuberculosis. One individual, one health agency or one Government cannot achieve the goal TB elimination by 2030. A global effort is the basic pre-requisite, incorporating advanced molecular diagnosis and appropriate treatment would ensure achieving a TB free world by 2030.

Carbapenem-resistant biofilm-producing multidrug resistant Klebsiella pneumoniae induced hospital-acquired infections and analysis of resistant genes

In recent years, drug resistance has caused a significant number of fatalities. This review focuses on the emergence of medication resistance in Klebsiella pneumoniae strains associated with biofilm development, neonatal sepsis, aging, and secondary K. pneumoniae infection in COVID-19 cases. This review is an exclusive survey of >200 research articles and >100 review articles indexed in the Web of Science list with the keywords “K. pneumoniae,” “drug resistance,” “sepsis,” “neonatal sepsis,” “sepsis in older age,” “secondary infection,” “biofilm production,” “secondary infection in COVID-19,” “drug resistance genes,” and “whole genome sequencing”. In order to protect antibiotic susceptibility and to provide an overview of the biofilm forming mechanisms among gram-negative bacteria, the review discusses the function of multidrug-resistant K. pneumoniae in the production of biofilms. Additionally, K. pneumoniae’s role in contemporary cases of sepsis, spread of medication resistance in hospitals, and sepsis caused by K. pneumoniae in the geriatric age group are discussed. A summary of current approaches and the identification of drug resistance genes utilizing a whole genome sequencing technique are also reviewed in this article.

CHRONIC HEPATITIS C VIRUS INFECTION: IS IT STILL A CHALLENGE?

This review overlooks the current international, and national status of chronic hepatitis C. Presence of antiHCV antibodies is used as a marker for chronic hepatitis C. With the help of molecular biology techniques, seven major HCV genotypes were identified worldwide. In addition, the latest genome sequencing techniques (SBT, NextGeneration Sequencing) analyzed the relationship between the HLA genes and KIR genes with chronic hepatitis C. Three HLA alleles (HLA A*23:01, B*44:02, and C*04:02) and four KIR genes (KIR2DL3, KIR2DL5, KIR2DS4, and KIR3DL3) have been associated with the chronic HCV infection in Romania and worldwide.

Assessing the relative performance of fast molecular dating methods for phylogenomic data

Advances in genome sequencing techniques produced a significant growth of phylogenomic datasets. This massive amount of data represents a computational challenge for molecular dating with Bayesian approaches. Rapid molecular dating methods have been proposed over the last few decades to overcome these issues. However, a comparative evaluation of their relative performance on empirical data sets is lacking. We analyzed 23 empirical phylogenomic datasets to investigate the performance of two commonly employed fast dating methodologies: penalized likelihood (PL), implemented in treePL, and the relative rate framework (RRF), implemented in RelTime. They were compared to Bayesian analyses using the closest possible substitution models and calibration settings. We found that RRF was computationally faster and generally provided node age estimates statistically equivalent to Bayesian divergence times. PL time estimates consistently exhibited low levels of uncertainty. Overall, to approximate Bayesian approaches, RelTime is an efficient method with significantly lower computational demand, being more than 100 times faster than treePL. Thus, to alleviate the computational burden of Bayesian divergence time inference in the era of massive genomic data, molecular dating can be facilitated using the RRF, allowing evolutionary hypotheses to be tested more quickly and efficiently.

Prognostic and predictive biomarkers for immunotherapy in advanced renal cell carcinoma.

The therapeutic algorithm of renal cell carcinoma has been revolutionized by the approval of immunotherapy agents by regulatory agencies. However, objective and durable responses are still not observed in a large number of patients, and prognostic and predictive biomarkers for immunotherapy response are urgently needed. Prognostic models used in clinical practice are based on clinical and laboratory factors (such as hypercalcaemia, neutrophil count or Karnofsky Performance Status), but, with progress in molecular biology and genome sequencing techniques, new renal cell carcinoma molecular features that might improve disease course and outcomes prediction have been highlighted. An implementation of current models is needed to improve the accuracy of prognosis in the immuno-oncology era. Moreover, several potential biomarkers are currently under evaluation, but effective markers to select patients who might benefit from immunotherapy and to guide therapeutic strategies are still far from validation.

A Whole Genome Sequencing-Based Approach to Track down Genomic Variants in Itraconazole-Resistant Species of Aspergillus from Iran.

The antifungal resistance in non-fumigatus Aspergillus spp., as well as Aspergillus fumigatus, poses a major therapeutic challenge which affects the entire healthcare community. Mutation occurrence of cyp51 gene paralogs is the major cause of azole resistance in Aspergillus spp. To obtain a full map of genomic changes, an accurate scan of the entire length of the Aspergillus genome is necessary. In this study, using whole genome sequencing (WGS) technique, we evaluated the mutation in cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA genes in different clinical isolates of Aspergillus fumigatus, Aspergillus niger, Aspergillus tubingensis, Aspergillus welwitschiae and Aspergillus terreus which responded to minimum inhibitory concentrations of itraconazole above 16 µg mL−1. We found different nonsynonymous mutations in the cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA gene loci. According to our findings, Aspergillus species isolated from different parts of the world may represent different pattern of resistance mechanisms which may be revealed by WGS.

Erythrina stem borer moth in California - New taxonomic status and implications for control of this emerging pest.

During the last 10 years, the Erythrina stem borer moth, Terastia meticulosalis, emerged as a pest of cultivated coral trees (Erythrina spp.) in California. Erythrina trees are valued for their moderate drought resistance and beautiful flame-like flowers. They are beloved enough to be considered Los Angeles's official "City Tree." Thus, they are a valuable horticultural crop and are grown by many nurseries and occur throughout the landscape in coastal southern California. Coral trees have been heavily affected by T. meticulosalis recently. Using whole genome sequencing techniques, we analysed the origins of this and other infestations of Erythrina in coastal areas and found that they have likely originated from the repeated expansions of the native range of the species in Arizona, a process possibly driven by climatic factors and/or movement of plants by humans. We also found sufficient genetic differences between the western population of the moth and the rest of the New World populations to describe a new western subspecies, T. meticulosalis occidentalis Sourakov & Grishin ssp. n. (type locality USA: CA, San Diego Co., La Jolla). These findings are of economic importance for future attempts to control the moth's impact on activities surrounding the horticultural use of Erythrina spp. by the Californian landscape and nursery industries.

A convergent mechanism of sex determination in dioecious plants: Distinct sex-determining genes display converged regulation on floral B-class genes.

Sex determination in dioecious plants has been broadly and progressively studied with the blooming of genome sequencing and editing techniques. This provides us with a great opportunity to explore the evolution and genetic mechanisms underlining the sex-determining system in dioecious plants. In this study, comprehensively reviewing advances in sex-chromosomes, sex-determining genes, and floral MADS-box genes in dioecious plants, we proposed a convergent model that governs plant dioecy across divergent species using a cascade regulation pathway connecting sex-determining genes and MADS-box genes e.g., B-class genes. We believe that this convergent mechanism of sex determination in dioecious plants will shed light on our understanding of gene regulation and evolution of plant dioecy. Perspectives concerning the evolutionary pathway of plant dioecy are also suggested.

PGT for structural chromosomal rearrangements in 300 couples reveals specific risk factors but an interchromosomal effect is unlikely

Research questionWhat factors affect the proportion of chromosomally balanced embryos in structural rearrangement carriers? Is there any evidence for an interchromosomal effect (ICE)? DesignPreimplantation genetic testing outcomes of 300 couples (198 reciprocal, 60 Robertsonian, 31 inversion and 11 complex structural rearrangement carriers) were assessed retrospectively. Blastocysts were analysed either by array-comparative genomic hybridization or next-generation sequencing techniques. ICE was investigated using a matched control group and sophisticated statistical measurement of effect size (φ). Results300 couples underwent 443 cycles; 1835 embryos were analysed and 23.8% were diagnosed as both normal/balanced and euploid. The overall cumulative clinical pregnancy and live birth rates were 69.5% and 55.8%, respectively. Complex translocations and female age (≥35) were found to be risk factors associated with lower chance of having a transferable embryo (P < 0.001). Based on analysis of 5237 embryos, the cumulative de-novo aneuploidy rate was lower in carriers compared to controls (45.6% versus 53.4%, P < 0.001) but this was a ‘negligible’ association (φ < 0.1). A further assessment of 117,033 chromosomal pairs revealed a higher individual chromosome error rate in embryos of carriers compared to controls (5.3% versus 4.9%), which was also a ‘negligible’ association (φ < 0.1), despite a P-value of 0.007. ConclusionsThese findings suggest that rearrangement type, female age and sex of the carrier have significant impacts on the proportion of transferable embryos. Careful examination of structural rearrangement carriers and controls indicated little or no evidence for an ICE. This study helps to provide a statistical model for investigating ICE and an improved personalized reproductive genetics assessment for structural rearrangement carriers.

Potentials and challenges of chromosomal microarray analysis in prenatal diagnosis.

Introduction: For decades, conventional karyotyping analysis has been the gold standard for detecting chromosomal abnormalities during prenatal diagnosis. With the development of molecular cytogenetic methods, this situation has dramatically changed. Chromosomal microarray analysis (CMA), a method of genome-wide detection with high resolution, has been recommended as a first-tier test for prenatal diagnosis, especially for fetuses with structural abnormalities. Methods: Based on the primary literature, this review provides an updated summary of the application of CMA for prenatal diagnosis. In addition, this review addresses the challenges that CMA faces with the emergence of genome sequencing techniques, such as copy number variation sequencing, genome-wide cell-free DNA testing, and whole exome sequencing. Conclusion: The CMA platform is still suggested as priority testing methodology in the prenatal setting currently. However, pregnant women may benefit from genome sequencing, which enables the simultaneous detection of copy number variations, regions of homozygosity and single-nucleotide variations, in near future.

GpemDB: A Scalable Database Architecture with the Multi-omics Entity-relationship Model to Integrate Heterogeneous Big-data for Precise Crop Breeding.

With the development of high-throughput genome sequencing and phenotype screening techniques, there is a possibility of leveraging multi-omics to speed up the breeding process. However, the heterogeneity of big data handicaps the progress and the lack of a comprehensive database supporting end-to-end association analysis impedes the efficient use of these data. In response to this problem, a scalable entity-relationship model and a database architecture are firstly proposed in this paper to manage the cross-platform data sets and explore the relationship among multi-omics, and finally accelerate our breeding efficiency. First, the targeted omics data of crops should be normalized before being stored in the database. A typical breeding data content and structure is demonstrated with the case study of rice (Oryza sativa L). Second, the structure, patterns and hierarchy of multi-omics data are described with the entity-relationship modeling technique. Third, some statistical tools used frequently in the agricultural analysis have been embedded into the database to help breeding. As a result, a general-purpose scalable database, called GpemDB integrating genomics, phenomics, enviromics and management, is developed. It is the first database designed to manage all these four omics data together. The GpemDB involving Gpem metadata-level layer and informative-level layer provides a visualized scheme to display the content of the database and facilitates users to manage, analyze and share breeding data. GpemDB has been successfully applied to a rice population, which demonstrates this database architecture and model are promising to serve as a powerful tool to utilize the big data for high precise and efficient research and breeding of crops.

Streptomyces sp. AC04842: Genomic Insights and Functional Expression of Its Latex Clearing Protein Genes (lcp1 and lcp2) When Cultivated With Natural and Vulcanized Rubber as the Sole Carbon Source.

Rubber-degrading Actinobacteria have been discovered and investigated since 1985. Only recently, through the advancement of genomic sequencing and molecular techniques, genes and pathways involved in rubber degradation are being revealed; however, the complete degradation pathway remains unknown. Streptomyces sp. AC04842 (JCM 34241) was discovered by screening at a Culture Collection Centre in Sarawak for Actinomycetes forming a clear zone on natural rubber latex agar. Streptomyces is a dominant and well-studied soil bacterium playing an important role in soil ecology including carbon recycling and biodegradation. Streptomyces sp. AC04842 draft genome revealed the presence of 2 putative latex clearing protein (lcp) genes on its chromosome and is closely related to Streptomyces cellulosae. Under the Streptomyces genus, there are a total of 64 putative lcp genes deposited in the GenBank and UniProt database. Only 1 lcp gene from Streptomyces sp. K30 has been characterized. Unlike Streptomyces sp. K30 which contained 1 lcp gene on its chromosome, Streptomyces sp. AC04842 contained 2 lcp genes on its chromosome. Streptomyces sp. AC04842 lcp1 and lcp2 amino acid sequences showed 46.13 and 69.11%, respectively, similarity to lcp sequences of Streptomyces sp. K30. Most rubber degrading strains were known to harbor only 1 lcp gene, and only recently, 2–3 lcp homologs have been reported. Several studies have shown that lcp-homolog expression increased in the presence of rubber. To study the expression of lcp1 and lcp2 genes for Streptomyces sp. AC04842, the strain was incubated in different types of rubber as the sole carbon source. In general, the lcp1 gene was highly expressed, while the lcp2 gene expression was upregulated in the presence of vulcanized rubber. Mixtures of natural and vulcanized rubber did not further increase the expression of both lcp genes compared with the presence of a specific rubber type. In this study, we paved the way to the exploration of lcp homologs and their function in degrading different types of rubber.

Recent progress of circular RNAs in different types of human cancer: Technological landscape, clinical opportunities and challenges (Review).

Circular RNAs (circRNAs) are a novel class of endogenous non‑coding RNAs that have been recently regarded as functionally active. CircRNAs are remarkably stable and known to possess several biological functions such as microRNA sponging, regulating transcription and splicing and occasionally acting as polypeptide‑producing templates. CircRNAs show tissue‑specific expression and have been reported to be associated with the progression of several types of malignancies. Given the recent progress in genome sequencing and bioinformatics techniques, a rapid increment in the biological role of circRNAs has been observed. Concurrently, the patent search from different patent databases shows that the patent number of circRNA is increasing very quickly. These phenomena reveal a rapid development of the technological landscape. In the present review, the recent progress on circRNAs in various kinds of cancer has been investigated and their function as biomarkers or therapeutic targets and their technological landscape have been appreciated. A new insight into circRNAs structure and functional capabilities in cancer has been reviewed. Continually increasing knowledge on their critical role during cancer progression is projecting them as biomarkers or therapeutic targets for various kinds of cancer. Thus, recent updates on the functional role of circRNAs in terms of the technological landscape, clinical opportunities (biomarkers and therapeutic targets), and challenges in cancer have been illustrated.

Genetic load: genomic estimates and applications in non-model animals.

Genetic variation, which is generated by mutation, recombination and gene flow, can reduce the mean fitness of a population, both now and in the future. This 'genetic load' has been estimated in a wide range of animal taxa using various approaches. Advances in genome sequencing and computational techniques now enable us to estimate the genetic load in populations and individuals without direct fitness estimates. Here, we review the classic and contemporary literature of genetic load. We describe approaches to quantify the genetic load in whole-genome sequence data based on evolutionary conservation and annotations. We show that splitting the load into its two components - the realized load (or expressed load) and the masked load (or inbreeding load) - can improve our understanding of the population genetics of deleterious mutations.

A robust association test with multiple genetic variants and covariates.

Due to the advancement of genome sequencing techniques, a great stride has been made in exome sequencing such that the association study between disease and genetic variants has become feasible. Some powerful and well-known association tests have been proposed to test the association between a group of genes and the disease of interest. However, some challenges still remain, in particular, many factors can affect the performance of testing power, e.g., the sample size, the number of causal and non-causal variants, and direction of the effect of causal variants. Recently, a powerful test, called TREM , is derived based on a random effects model. TREM has the advantages of being less sensitive to the inclusion of non-causal rare variants or low effect common variants or the presence of missing genotypes. However, the testing power of TREM can be low when a portion of causal variants has effects in opposite directions. To improve the drawback of TREM , we propose a novel test, called TROB , which keeps the advantages of TREM and is more robust than TREM in terms of having adequate power in the case of variants with opposite directions of effect. Simulation results show that TROB has a stable type I error rate and outperforms TREM when the proportion of risk variants decreases to a certain level and its advantage over TREM increases as the proportion decreases. Furthermore, TROB outperforms several other competing tests in most scenarios. The proposed methodology is illustrated using the Shanghai Breast Cancer Study.

Cytoskeletal Protein Variants Driving Atrial Fibrillation: Potential Mechanisms of Action.

The most common clinical tachyarrhythmia, atrial fibrillation (AF), is present in 1–2% of the population. Although common risk factors, including hypertension, diabetes, and obesity, frequently underlie AF onset, it has been recognized that in 15% of the AF population, AF is familial. In these families, genome and exome sequencing techniques identified variants in the non-coding genome (i.e., variant regulatory elements), genes encoding ion channels, as well as genes encoding cytoskeletal (-associated) proteins. Cytoskeletal protein variants include variants in desmin, lamin A/C, titin, myosin heavy and light chain, junctophilin, nucleoporin, nesprin, and filamin C. These cytoskeletal protein variants have a strong association with the development of cardiomyopathy. Interestingly, AF onset is often represented as the initial manifestation of cardiac disease, sometimes even preceding cardiomyopathy by several years. Although emerging research findings reveal cytoskeletal protein variants to disrupt the cardiomyocyte structure and trigger DNA damage, exploration of the pathophysiological mechanisms of genetic AF is still in its infancy. In this review, we provide an overview of cytoskeletal (-associated) gene variants that relate to genetic AF and highlight potential pathophysiological pathways that drive this arrhythmia.

Use of Circulating Tumour DNA to Assess Minimal Residual Disease in Gastrointestinal Cancers

Despite our modern perioperative therapies, many patients with gastrointestinal cancer relapse after surgery. Novel strategies to identify and treat patients at high risk of relapse are needed to improve cancer outcomes. Circulating tumour DNA (ctDNA) is a promising, non-invasive biomarker with the potential to identify the earliest signs of cancer relapse. The presence of tumourspecific DNA in the blood in the absence of visualized tumour is suggestive of minimal residual disease and forebodes measurable relapse. Genomic sequencing techniques have advanced over the past few decades, and we have become better able to detect significantly low levels of DNA circulating in the blood from low-volume disease. Numerous studies using various technologies have established ctDNA as a powerful prognostic biomarker for relapse and survival in gastrointestinal cancers. ctDNA has the potential to risk-stratify patients in the postoperative, post-adjuvant and longitudinal settings for therapeutic escalation or de-escalation strategies. It may also capture early tumour dynamics in response to therapeutic intervention. As the multifaceted potential of ctDNA is attracting the attention of researchers, clinicians and patients, many questions remain regarding its use, interpretation and limitations. Here, we discuss the current understanding of ctDNA for minimal residual disease evaluation in gastrointestinal cancers and potential future directions.