Next-generation Sequencing Techniques Research Articles

Towards enhancing the user experience of ChIP-Seq data analysis web tools

<p>Deoxyribonucleic acid (DNA) sequencing is the process of locating the sequence of the main chemical bases in the DNA. Next-generation sequencing (NGS) is the state-of-the-art DNA sequencing technique. The NGS technique advanced the biological science in analyzing human DNA due to its scalability, high throughput, and speed. Analyzing human DNA is crucial to determine the ability of a person to develop certain diseases and his ability to respond to certain medications. ChIP-sequencing is a method that combines chromatin immunoprecipitation (ChIP) with NGS sequencing to analyze protein interactions with DNA to identify binding sites. Many online web tools have been developed to conduct ChIP-Seq data analysis to either discover or find motifs, i.e., patterns of binding sites. Since these ChIP-Seq web tools need to be used by clinical practitioners, they must comply to the web-related usability tasks including effectiveness, efficiency and satisfaction to enhance the user experience (UX). To that end, we have conducted an empirical study to understand their UX design. Specifically, we have evaluated the usability of 8 widely used ChIP-Seq web tools against 6 known usability quality metrics. Our study shows that the design of the studied ChIP-Seq web tools does not follow the UX design principles.</p>

Implications of Microorganisms in Alzheimer’s Disease

Alzheimer’s disease (AD) is a deadly brain degenerative disorder that leads to brain shrinkage and dementia. AD is manifested with hyperphosphorylated tau protein levels and amyloid beta (Aβ) peptide buildup in the hippocampus and cortex regions of the brain. The nervous tissue of AD patients also contains fungal proteins and DNA which are linked to bacterial infections, suggesting that polymicrobial infections also occur in the brains of those with AD. Both immunohistochemistry and next-generation sequencing (NGS) techniques were employed to assess fungal and bacterial infections in the brain tissue of AD patients and non-AD controls, with the most prevalent fungus genera detected in AD patients being Alternaria, Botrytis, Candida, and Malassezia. Interestingly, Fusarium was the most common genus detected in the control group. Both AD patients and controls were also detectable for Proteobacteria, followed by Firmicutes, Actinobacteria, and Bacteroides for bacterial infection. At the family level, Burkholderiaceae and Staphylococcaceae exhibited higher levels in the brains of those with AD than the brains of the control group. Accordingly, there is thought to be a viscous cycle of uncontrolled neuroinflammation and neurodegeneration in the brain, caused by agents such as the herpes simplex virus type 1 (HSV1), Chlamydophila pneumonia, and Spirochetes, and the presence of apolipoprotein E4 (APOE4), which is associated with an increased proinflammatory response in the immune system. Systemic proinflammatory cytokines are produced by microorganisms such as Cytomegalovirus, Helicobacter pylori, and those related to periodontal infections. These can then cross the blood–brain barrier (BBB) and lead to the onset of dementia. Here, we reviewed the relationship between the etiology of AD and microorganisms (such as bacterial pathogens, Herpesviridae viruses, and periodontal pathogens) according to the evidence available to understand the pathogenesis of AD. These findings might guide a targeted anti-inflammatory therapeutic approach to AD.

Keystone Taxa and Predictive Functional Analysis of Sphagnum palustre Tank Microbiomes in Erxianyan Peatland, Central China.

Simple SummaryDeciphering the relationship between microbiome of keystone species Sphagnum palustre and potential function in the Erxianyan peatland ecosystems is important in the context of global peatland degradation. We evaluated the S. palustre tank microbiome and predicted the potential ecological functions. In total, 38 phyla, 55 classes, 122 orders and 490 genera were detected. Proteobacteria and Firmicutes are the dominant endophytes in S. palustre. Core microbiomes are mainly found in 7 phyla, 9 classes, 15 orders, 22 families and 42 genera. Functions predictive of microbial communities are involved in nitrogen fixation, carbon cycle, nitrate metabolism, sulfate respiration and chitinolysis, which may enable the Sphagnum to adapt to harsh environmental conditions. This study provides new insights into the relationship between Sphagnum-associated microbiomes and their potential ecological functions in subalpine peatlands.Sphagnum is a fundamental ecosystem of engineers, including more than 300 species around the world. These species host diverse microbes, either endosymbiotic or ectosymbiotic, and are key to carbon sequestration in peatland ecosystems. However, the linkages between different types of Sphagnum and the diversity and ecological functions of Sphagnum-associated microbiomes are poorly known, and so are their joint responses to ecological functions. Here, we systematically investigated endophytes in Sphagnum palustre via next-generation sequencing (NGS) techniques in the Erxianyan peatland, central China. The total bacterial microbiome was classified into 38 phyla and 55 classes, 122 orders and 490 genera. The top 8 phyla of Proteobacteria (33.69%), Firmicutes (11.94%), Bacteroidetes (9.42%), Actinobacteria (6.53%), Planctomycetes (6.37%), Gemmatimonadetes (3.05%), Acidobacteria (5.59%) and Cyanobacteria (1.71%) occupied 78.31% of total OTUs. The core microbiome of S. palustre was mainly distributed mainly in 7 phyla, 9 classes, 15 orders, 22 families and 43 known genera. There were many differences in core microbiomes compared to those in the common higher plants. We further demonstrate that the abundant functional groups have a substantial potential for nitrogen fixation, carbon cycle, nitrate metabolism, sulfate respiration and chitinolysis. These results indicate that potential ecological function of Sphagnum palustre in peatlands is partially rooted in its microbiomes, and that incorporating into functional groups of Sphagnum-associated microbiomes can promote mechanistic understanding of Sphagnum ecology in subalpine peatlands.

Identifying Human Remains from 20th Century Warfare: A State of the Field Essay.

As we continually reflect on the wars of the 20th century, identification of the remains of victims takes an increasingly prominent position in ongoing research. Existing work on the identification of human remains from 20th century wars primarily covers the determination of phenotypic characteristics, kinship and geographic origins, supporting the establishment of genetic information databases. Compared with standard forensic methods, DNA analyses have revealed greater effectiveness. The process of DNA analysis includes DNA extraction, genetic marker testing and data analysis. Protocols from ancient DNA research can be applied to degraded remains, and next-generation sequencing (NGS) techniques can compensate for shortcomings in the most commonly-used PCR-capillary electrophoresis typing. As it stands, wide-ranging inter-governmental and inter-institutional collaboration is necessary in order to set up NGS-based public databases, and thereby promote the identification of human remains and archaeological forensics.

Literature review on genotype-phenotype correlation in patients with hereditary spherocytosis.

Hereditary spherocytosis (HS) is a prevalent inherited hemolytic disorder primarily reported in Caucasians. Recently, next-generation sequencing (NGS) techniques have shown tremendous potential in the diagnosis of HS. HS commonly originates from variants in ANK1, SPTB, SLC4A1, SPTA1, and EPB42. This review is focused on 13 previous clinical studies on genotype-phenotype correlation, which might promote the role of causative variants in the diagnosis and prognosis of HS. Most studies have focused on the pediatric population and Asian countries. The occurrence of novel variants was common in each cohort, and variants with a high frequency of causative genes were demonstrated. In conclusion, patients with variants in SPTA1 and SLC4A1 were reported to have more severe and milder anemia, respectively. ANK1 and SPTB are the most common variants in patients with HS, and no significant difference in phenotypes was observed between patients with variants in ANK1 versus SPTB. The types and locations of variants might influence the phenotype of each genotype, whereas the roles of concomitant pathogenic genes and the source of variants deserve further investigation.

Differential transcriptome profile underlying risky choice in a rat gambling task.

Proper measurement of expected risk is important for making rational decisions, and maladaptive decision making may underlie various psychiatric disorders. However, differentially expressed genetic profiling involved in this process is still largely unknown. A rodent version of the gambling task (rGT) has been developed to measure decision-making by adopting the same principle of Iowa Gambling Task in humans. In the present study, we examined using next-generation sequencing (NGS) technique whether there are differences in gene expression profiles in the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc) when rats make different choices toward risk in rGT. Rats were trained in a touch screen chamber to learn the relationships between 4 different light signals on the window of the screen and accompanied reward outcomes or punishments set up with different magnitudes and probabilities. Once they showed a stabilized pattern of preference upon free choice, rats were classified into risk-averse or risk-seeking groups. After performing the rGT, rats were decapitated, the mPFC and the NAc was dissected out, and NGS was performed with the total RNA extracted. We found that 477 and 36 genes were differentially expressed (approximately 75 and 83% out of them were downregulated) in the mPFC and the NAc, respectively, in risk-seeking compared to risk-averse rats. Among those, we suggested a few top ranked genes that may contribute to promoting risky choices. Our findings provide insights into transcriptional components underlying risky choices in rats.

Research progress and clinical application of the third- generation sequencing techniques

The Sanger sequencing techniques, also known as the first-generation sequencing techniques and the gold standard of sequencing, have promoted the completion of "working draft" of the human genome, but the disadvantages of low throughput and high cost limit its large-scale application. The second-generation sequencing techniques, also known as the next-generation sequencing techniques, have widely used in basic research and clinical application because of its high throughput and low cost, but the short reads has always been an unavoidable shortcoming. Then, the emergence of the third-generation sequencing techniques, with the long reads, provides new technology selection for the analysis of complex repetitive regions on genome sequences and the assembly of high-quality genomes. In recent years, the third-generation sequencing techniques have been further developed, and have gradually demonstrated the clinical application value. This article reviewed the research progress and clinical application of the third-generation sequencing techniques.

Rapid genome sequencing for pediatrics.

The advancements made in next-generation sequencing (NGS) technology over the past two decades have transformed our understanding of genetic variation in humans and had a profound impact on our ability to diagnose patients with rare genetic diseases. In this review, we discuss the recently developed application of rapid NGS techniques, used to diagnose pediatric patients with suspected rare diseases who are critically ill. We highlight the challenges associated with performing such clinical diagnostics tests in terms of the laboratory infrastructure, bioinformatic analysis pipelines, and the ethical considerations that need to be addressed. We end by looking at what future developments in this field may look like and how they can be used to augment the genetic data to further improve the diagnostic rates for these high-priority patients.

Fluctuations in Intestinal Microbiota Following Ingestion of Natto Powder Containing Bacillus subtilis var. natto SONOMONO Spores: Considerations Using a Large-Scale Intestinal Microflora Database.

Improving the intestinal microbiota using probiotics, prebiotics, and synbiotics has attracted attention as a method of disease prevention and treatment. This is the first study to discuss the effects of food intake on the intestinal microbiota using a large Japanese intestinal microbiota database. Here, as a case study, we determined changes in the intestinal microbiota caused by ingestion of a processed natto food containing B. subtilis var. natto SONOMONO spores, SONOMONO NATTO POWDER CAPSULESTM, by analyzing 16S rRNA sequence data generated using next-generation sequencing techniques. The results showed that the relative abundance of Bifidobacterium and Blautia as well as the relative abundance of Bifidobacterium were increased in males and females in the ingesting group, respectively. Additionally, the effects of SONOMONO NATTO POWDER CAPSULESTM intake on Bifidobacterium and Blautia abundance depended on the relative abundance of Bifidobacterium at baseline. Finally, analysis of a large Japanese intestinal microbiota database suggested that the bacterial genera that fluctuated with the ingestion of SONOMONO NATTO POWDER CAPSULESTM may be associated with lifestyle-related diseases such as diabetes.

Effectiveness of Whole-Exome Sequencing for the Identification of Causal Mutations in Patients with Suspected Inherited Ocular Diseases.

Genetic eye disorders, affecting around one in 1000 people, encompass a diverse group of diseases causing severe visual deficiency. The recent adoption of next-generation sequencing techniques, including whole-exome sequencing (WES), in medicine has greatly enhanced diagnostic rates of genetically heterogeneous diseases. The objectives of the study were to assess the diagnostic yield of WES in a cohort of Mexican individuals with suspected genetic eye disorders and to evaluate the improvement of diagnostic rates by reanalysis of WES data in patients without an initial molecular diagnosis. A total of 90 probands with ocular anomalies of suspected genetic origin were ascertained. Patients underwent WES in leukocytic DNA. Bioinformatics analysis and Sanger sequencing were used to confirm the disease-causing variants. Only variants identified as pathogenic or likely pathogenic were considered as causal. Initial analysis revealed causal mutations in 46 cases (51%). Reanalysis of WES data 12 months after first analysis resulted in the identification of additional causal variants in 6 patients (7%), increasing the molecular diagnostic yield to 58%. The highest diagnostic rates by disease categories corresponded to hereditary retinal dystrophies (77%) and to anomalies of the anterior segment of the eye (47%). Our study demonstrates that WES is an effective approach for genetic diagnosis of genetic ocular diseases and that reanalysis of WES data can improve the diagnostic yield.

Perspective on the use of synthetic biology in rudimentary food fermentations

Rudimentary food fermentation can be defined as a spontaneous process of conversion of food components through enzymatic action. A great variety of fermented foods are produced using spontaneous approaches; however, cocoa and coffee represent the most important agricultural commodities on international markets. As a manner to increase the efficiency of these processes, starter cultures have been developed and applied under field conditions. The selection process begins with the recovery of microbial strains from spontaneous fermentation through phenotypic and metabolic traits. Next, mutation-based breeding is used to develop and improve well-adapted starter cultures. With advances in synthetic biology, especially in the last decade, the development of robust cellular fabrications with high fermentative capacity has become easier—largely due to the development of genomic approaches, such as next-generation sequencing techniques, CRISPR-Cas system and bioinformatics tools. This review brings prospects on the use of synthetic biology to design new robust strains for use in cocoa and coffee fermentations, but which can be extended to other rudimentary foods. In addition, metabolic traits and target genes (e.g., UvrA, RecA, GPD1, and GPP2) are proposed as a starting point for the improvement of cocoa and coffee starters. Finally, the regulatory and safety requirements for these food crops are addressed. This review aims to stimulate research on the process of fermentation and the associated synthetic biology tools to produce fermented food efficiently and sustainably.

A comprehensive review and meta-analysis of recent advances in biotechnology for plant virus research and significant accomplishments in human health and the pharmaceutical industry

ABSTRACT Secondary metabolites made by plants and used through their metabolic routes are today’s most reliable and cost-effective way to make pharmaceuticals and improve health. The concept of genetic engineering is used for molecular pharming. As more people use plants as sources of nanotechnology systems, they are adding to this. These systems are made up of viruses-like particles (VLPs) and virus nanoparticles (VNPs). Due to their superior ability to be used as plant virus expression vectors, plant viruses are becoming more popular in pharmaceuticals. This has opened the door for them to be used in research, such as the production of medicinal peptides, antibodies, and other heterologous protein complexes. This is because biotechnological approaches have been linked with new bioinformatics tools. Because of the rise of high-throughput sequencing (HTS) and next-generation sequencing (NGS) techniques, it has become easier to use metagenomic studies to look for plant virus genomes that could be used in pharmaceutical research. A look at how bioinformatics can be used in pharmaceutical research is also covered in this article. It also talks about plant viruses and how new biotechnological tools and procedures have made progress in the field.

Detection of Novel Candidate Mutations as A Cause of Steroid-resistant Nephrotic Syndrome in Children using Next-generation Sequencing Techniques

Detection of Novel Candidate Mutations as A Cause of Steroid-resistant Nephrotic Syndrome in Children using Next-generation Sequencing Techniques

EP16.01-007 Molecular Characterization by Next-Generation Sequencing (NGS) of Patients with Non-Small Cell Lung Cancer (NSCLC) Treated with Immunotherapy

Immunotherapy has transformed the treatment of non-small cell lung cancer (NSCLC). On the other hand, the incorporation of Next-Generation Sequencing (NGS) techniques can provide great information at the molecular level. The aim of our study is to integrate the clinical and molecular information of a cohort of patients treated with immunotherapy to define molecular subtypes.

216.17: Multiplex Digital PCR for the Detection of Donor-derived Cell-free DNA

Introduction: Graft damage and failure are major problems after solid-organ transplantation. Biomarkers for monitoring transplanted patients provide new opportunities to substantially improve patient management, graft lifespan, and patient survival. There is strong evidence that donor-derived cell-free DNA (DD-cfDNA) increases when transplanted organs are damaged. The majority of this information is derived from tests using next-generation sequencing. Here we present an evaluation of new technology, multiplex digital PCR (mdPCR), which has the potential to revolutionize DD-cfDNA testing. Method: This mdPCR method takes advantage of Luminex’s DigiMAP multiplex PCR chemistry and the QuantStudio Absolute Q Digital PCR platform (Thermo Fisher Scientific, previously Combinati). The bench work for mdPCR is completed in less than five hours. Data is analyzed using a custom R package. Assay performance characteristics were determined using artificial mixtures of genomic DNA, and inter-laboratory reproducibility was assessed. Blood was collected from kidney and heart transplant recipients, cf-DNA was isolated using a variety of methods, and mdPCR was used to quantify DD-cfDNA levels. Results: This methodology distinguished donor and recipient DNA and provided absolute quantification of the number of DNA copies. The following performance characteristics were determined: sensitivity, the limit of detection, and linear range, inter- and intra- assay consistency. Several factors affected performance including: DNA characteristics, probe design, primer design, and PCR reaction components. Results showed that high levels of DD-cfDNA are consistent with clinical observations and that rapid testing is feasible (< five hours). Conclusion: The mdPCR approach has high promise to revolutionize diagnostic testing in cancer, infectious disease, and transplant monitoring. Our study demonstrates the advantages of this technology for the detection of DD-cfDNA which includes absolute quantitation, sensitivity, ease of use, short turn-around-time, and a relatively low cost when compared to next-generation sequencing techniques. A test with these features could transform the management of transplant recipients and lead to prolonged graft and patient survival.

A Novel Mutation in the TSEN2 Gene Among Two Iranian Families with Pontocerebellar Hypoplasia Type 2B

Background: Pontocerebellar hypoplasia is an autosomal recessive and neurodegenerative disorder divided into sixteen subtypes. Pontocerebellar hypoplasia type 2B (PCH2B) shows microcephaly combined with epilepsy extrapyramidal dyskinesia and chorea due to different homozygous or compound heterozygous pathogenic mutations in the TSEN2 gene. Objectives: This study was aimed to find the mutation responsible for pontocerebellar hypoplasia in two families with similar patients using the next-generation sequencing technique. Methods: Whole exome sequencing (WES) was performed for two individuals in families with microcephaly and developmental delay. A novel mutation was confirmed by Sanger sequencing and segregation analysis. Pathogenicity and the effect of mutation were investigated using different bioinformatics analyses. Results: WES showed a novel and potential pathogenic mutation in TSEN2 (p.R350Q: c.1049G&gt;A). Sanger sequencing verified that both individuals were homozygote and that their parents were heterozygote for this variant. Conclusions: Our data can help enhance knowledge about the role of the TSEN2 gene in pontocerebellar hypoplasia. Also, our results can extend the list of mutations responsible for PCH2B and help find the correlation between the genotypes and phenotypes for this disorder.

Next-Generation Sequencing of Local Romanian Tomato Varieties and Bioinformatics Analysis of the Ve Locus.

Genetic variability is extremely important, not only for the species’ adaptation to environmental challenges, but also for the creation of novel varieties through plant breeding. Tomato is an important vegetable crop, as well as a model species in numerous genomic studies. Its genome was fully sequenced in 2012 for the ‘Heinz 1706’ variety, and since then, resequencing efforts have revealed genetic variability data that can be used for multiple purposes, including triggering mechanisms of biotic and abiotic stress resistance. The present study focused on the analysis of the genome variation for eight Romanian local tomato varieties using next-generation sequencing technique, and as a case study, the sequence analysis of the Ve1 and Ve2 loci, to determine which genotypes might be good candidates for future breeding of tomato varieties resistant to Verticillium species. The analysis of the Ve locus identified several genotypes that could be donors of the Ve1 gene conferring resistance to Verticillium race 1. Sequencing for the first time Romanian genotypes enriched the existing data on various world tomato genetic resources, but also opened the way for the molecular breeding in Romania. Plant breeders can use these data to create novel tomato varieties adapted to the ever-changing environment.

European and American chestnuts: An overview of the main threats and control efforts.

Chestnuts are multipurpose trees significant for the economy and wildlife. These trees are currently found around the globe, demonstrating their genetic adaptation to different environmental conditions. Several biotic and abiotic stresses have challenged these species, contributing to the decline of European chestnut production and the functional extinction of the American chestnut. Several efforts started over the last century to understand the cellular, molecular, and genetic interactions behind all chestnut biotic and abiotic interactions. Most efforts have been toward breeding for the primary diseases, chestnut blight and ink disease caused by the pathogens, Cryphonectria parasitica and Phytophthora cinnamomi, respectively. In Europe and North America, researchers have been using the Asian chestnut species, which co-evolved with the pathogens, to introgress resistance genes into the susceptible species. Breeding woody trees has several limitations which can be mostly related to the long life cycles of these species and the big genome landscapes. Consequently, it takes decades to improve traits of interest, such as resistance to pathogens. Currently, the availability of genome sequences and next-generation sequencing techniques may provide new tools to help overcome most of the problems tree breeding is still facing. This review summarizes European and American chestnut’s main biotic stresses and discusses breeding and biotechnological efforts developed over the last decades, having ink disease and chestnut blight as the main focus. Climate change is a rising concern, and in this context, the adaptation of chestnuts to adverse environmental conditions is of extreme importance for chestnut production. Therefore, we also discuss the abiotic challenges on European chestnuts, where the response to abiotic stress at the genetic and molecular level has been explored.

Bioinformatics: From NGS Data to Biological Complexity in Variant Detection and Oncological Clinical Practice.

The use of next-generation sequencing (NGS) techniques for variant detection has become increasingly important in clinical research and in clinical practice in oncology. Many cancer patients are currently being treated in clinical practice or in clinical trials with drugs directed against specific genomic alterations. In this scenario, the development of reliable and reproducible bioinformatics tools is essential to derive information on the molecular characteristics of each patient’s tumor from the NGS data. The development of bioinformatics pipelines based on the use of machine learning and statistical methods is even more relevant for the determination of complex biomarkers. In this review, we describe some important technologies, computational algorithms and models that can be applied to NGS data from Whole Genome to Targeted Sequencing, to address the problem of finding complex cancer-associated biomarkers. In addition, we explore the future perspectives and challenges faced by bioinformatics for precision medicine both at a molecular and clinical level, with a focus on an emerging complex biomarker such as homologous recombination deficiency (HRD).

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

BackgroundMetastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC.MethodsA cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita’s overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher’s, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method.ResultsStratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03).ConclusionThese data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.