NGS Technology Research Articles

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada.

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.

Exploration of the MSI landscape in Chinese pan-cancer patient by Next-Generation Sequencing.

e14576 Background: Clinically, MSI had been used as an important molecular marker for the prognosis of colorectal cancer and other solid tumors and the formulation of adjuvant treatment plans, and it had been used to assist in the screening of Lynch syndrome. However, there were currently few reports on the incidence of MSI-H in Chinese pan-cancer patients. This study described the occurrence of MSI in a large multi-center pan-cancer cohort in China, and explored the correlation between MSI and patients' TMB, age, PD-L1 expression and other indicators. Methods: The study included 8361 patients with 8 cancer types from multiple tumor centers. Use immunohistochemistry to detect the expression of MMR protein (MLH1, MSH2, MSH6 and PMS2) in patients with various cancer types to determine the MSI status and detect the expression of PD-L1 in patients. Through NGS technology, 831 genes of 8361 Chinese cancer patients were sequenced and the tumor mutation load of the patients was calculated. The MSI mutations of patients in 8 cancer types were analyzed and the correlation between MSI mutations of patients and the patient's age, TMB and PD-L1 expression was analyzed. Results: The test results showed that MSI patients accounted for 1.66% of pan-cancers. Among them, MSI-H patients accounted for the highest proportion in intestinal cancer, reaching 7.2%. The correlation analysis between MSI and TMB was performed on patients of various cancer types. The results showed that: in each cancer type, MSI-H patients had TMB greater than 10, and 26.83% of MSI-H patients had TMB greater than 100 in colorectal cancer patients. The result of correlation analysis showed that there was no significant correlation between the patient's age and the risk of MSI mutation ( P> 0.05). In addition to PAAD and LUAD, the expression of PD-L1 in MSI-H patients was higher than that in MSS patients in other cancer types( P< 0.05). The correlation analysis between PD-L1 expression and TMB in patients found that in colorectal cancer, the higher the expression of PD-L1, the higher the patient's TMB ( P< 0.05). Conclusions: In this study, we explored the incidence of MSI-H in pan-cancer patients in China and found that the TMB was greater than 10 in patients with MSI-H. Compared with MSS patients, MSI-H patients have higher PD-L1 expression, and the higher the PD-L1 expression in colorectal cancer, the higher the TMB value of patients.

Why are oncologists not testing all NSCLC patients for biomarkers in the U.S. market?

e21003 Background: NSCLC has the greatest number of biomarkers of any tumor type. If a patient is not biomarker tested, he or she may not receive access to targeted therapies that often offer greater efficacy through personalized therapy. The purpose of this research study is to examine the reasons certain lung cancer patients are not tested in the United States for biomarkers. Methods: This study followed market research best practices. The study was based on a survey of the BrandImpact Oncology panel conducted on patient visits during Q4 2020 at the point-of-prescribing. Results: The baseline measure of NSCLC patients that were being treated but had a biomarker status that was unknown ranges from 9% to 12% of patient visits. The biomarkers examined included ALK, ROS-1, EGFR and PD-L1. The Oncologists who treat NSCLC patients received the following question: When treating patients in your practice with NSCLC cancer what are your top 3 reasons for not conducting biomarker testing? As outlined in the table below which reflects all survey responses, the top four reasons for not conducting biomarker testing are: not enough tumor sample for testing purposes, patient has early stage of disease, patient is not healthy enough and patient cost associated with testing. Conclusions: The overall results indicate the absence of biomarker testing for lung cancer patients is mainly due to two different patient types. The early-stage patient was a key reason for not testing despite advances in early stage indications and diagnostic technology which provide increasing evidence that testing should be done earlier in the treatment journey. The late-stage patient which is seen more often in Academic institutions and who often has more aggressive cancer. The speed of receiving the biomarker test results for these more severe patients likely needs to be addressed. In the Community, setting cost of branded therapies can be an issue and in some cases biomarker testing is not readily available in all practices. It should also be noted that “not enough tumor sample for testing” was the number one reason for not conducting biomarker testing, but this issue can now be addressed through liquid NGS technology.[Table: see text]

Research Based on Multimodal Deep Feature Fusion for the Auxiliary Diagnosis Model of Infectious Respiratory Diseases

Pulmonary infection is a common clinical respiratory tract infectious disease with a high incidence rate and a severe mortality rate as high as 30%–50%, which seriously threatens human life and health. Accurate and timely anti-infective treatment is the key to improving the cure rate. NGS technology provides a new, fast, and accurate method for pathogenic diagnosis, which can provide effective clues to the clinic, but determining the true pathogenic bacteria is a problem that needs to be solved urgently, and a comprehensive judgment must be made by the clinician combining the laboratory results, clinical information, and epidemiology. This paper intends to effectively collect and process the missing values of NGS data, clinical manifestations, laboratory test results, imaging test results, and other multimodal data of patients with infectious respiratory diseases. It also studies the deep feature fusion algorithm of multimodal data, couples the private and shared features of different modal data of infectious respiratory diseases, and digs into the hidden information of different modalities to obtain efficient and robust shared features that are conducive to auxiliary diagnosis. The establishment of an auxiliary diagnosis model for the infectious respiratory diseases can intelligentize and automate the diagnosis process of infectious respiratory, which has important significance and application value when applied to clinical practice.

The Genetics of Hereditary Angioedema: A Review.

Hereditary angioedema is a rare inherited disorder characterized by recurrent episodes of the accumulation of fluids outside of the blood vessels, causing rapid swelling of tissues in the hands, feet, limbs, face, intestinal tract, or airway. Mutations in SERPING1, the gene that encodes C1-INH (C1 esterase inhibitor), are responsible for the majority of cases of hereditary angioedema. C1 esterase inhibitor (C1-INH) is a major regulator of critical enzymes that are implicated in the cascades of bradykinin generation, which increases the vascular permeability and allows the flow of fluids into the extracellular space and results in angioedema. Moreover, a dominantly inherited disease has been described that has a similar clinical picture to C1-INH-HAE (Hereditary angioedema due to C1 inhibitor deficiency), but with normal C1-INH level and activity. This new type of HAE has no mutation in the SERPING1 gene and it is classified as nC1-INH-HAE (HAE with normal C1-INH). Currently mutations in six different genes have been identified as causing nC1-INH-HAE: factor XII (F12), plasminogen (PLG), angiopoietin 1 (ANGPT1), Kininogen 1 (KNG1), Myoferlin (MYOF), and heparan sulfate (HS)-glucosamine 3-O-sulfotransferase 6 (HS3ST6). In this review we aim to summarize the recent advances in genetic characterization of angioedema and possible future prospects in the identification of new genetic defects in HAE. We also provide an overview of diagnostic applications of genetic biomarkers using NGS technologies (Next Generation Sequencing).

Tremellomycetes Yeasts in Kernel Ecological Niche: Early Indicators of Enhanced Competitiveness of Endophytic and Mycoparasitic Symbionts against Wheat Pathobiota.

Tremellomycetes rDNA sequences previously detected in wheat kernels by MiSeq were not reliably assigned to a genus or clade. From comparisons of ribosomal internal transcribed spacer region (ITS) and subsequent phylogenetic analyses, the following three basidiomycetous yeasts were resolved and identified: Vishniacozyma victoriae, V. tephrensis, and an undescribed Vishniacozyma rDNA variant. The Vishniacozyma variant’s clade is evolutionarily close to, but phylogenetically distinct from, the V. carnescens clade. These three yeasts were discovered in wheat kernel samples from the Canadian prairies. Variations in relative Vishniacozyma species abundances coincided with altered wheat kernel weight, as well as host resistance to chemibiotrophic Tilletia (Common bunt—CB) and necrotrophic Fusarium (Fusarium head blight—FHB) pathogens. Wheat kernel weight was influenced by the coexistence of Vishniacozyma with endophytic plant growth-promoting and mycoparasitic biocontrol fungi that were acquired by plants. Kernels were coated with beneficial Penicillium endophyte and Sphaerodes mycoparasite, each of which had different influences on the wild yeast population. Its integral role in the kernel microbiome renders Vishniacozyma a measurable indicator of the microbiome–plant interaction. The ability of NGS technology to detect specific endophytic DNA variants and early changes in dynamics among symbionts within the kernel ecological niche enables the prediction of crop disease emergence, suggesting that advanced microbiological testing may be a potentially useful tool for both phytoprotection and more efficient wheat breeding programs.

Plant Microbiota Beyond Farming Practices: A Review

Plants have always grown and evolved surrounded by numerous microorganisms that inhabit their environment, later termed microbiota. To enhance food production, humankind has relied on various farming practices such as irrigation, tilling, fertilization, and pest and disease management. Over the past few years, studies have highlighted the impacts of such practices, not only in terms of plant health or yields but also on the microbial communities associated with plants, which have been investigated through microbiome studies. Because some microorganisms exert beneficial traits that improve plant growth and health, understanding how to modulate microbial communities will help in developing smart farming and favor plant growth-promoting (PGP) microorganisms. With tremendous cost cuts in NGS technologies, metagenomic approaches are now affordable and have been widely used to investigate crop-associated microbiomes. Being able to engineer microbial communities in ways that benefit crop health and growth will help decrease the number of chemical inputs required. Against this background, this review explores the impacts of agricultural practices on soil- and plant-associated microbiomes, focusing on plant growth-promoting microorganisms from a metagenomic perspective.

Exploring the use of new water quality indicators based on microbial communities

The growing concern for the quality of water in aquatic ecosystems makes it essential to develop new indicators that allow evaluating and predicting their state against anthropogenic impact. Microorganisms are able to reflect quickly changes in their habitat, through both its taxonomic and functional characteristics, and that is why they are in consideration as indicators of environmental quality (*1). The objective of this work was to identify attributes of the composition and functionality of microbial communities, to be evaluated as an indicator of water quality, focusing on emerging pollutants (ECs). For that, ECs and bacterial communities were analyzed along the basins of two coastal lagoons encompassing an anthropogenic gradient, looking for taxonomic and functional indicators. Taxonomic indicators were looked using Illumina sequencing of 16S RNAr gene V4 region followed by identification of amplicon secuence variants (ASVs) and taxonomic annotation. In the case of functional indicators, shotgun sequencing was used added to identification and annotation of open reading frames (ORFs). Clustering techniques were implemented to define groups of sites based on the concentration of different categories of ECs. Then, the indicator value analysis (IndVal) (*2) was performed to identify taxonomic and functional traits that could be used as indicators of those groups. Finally, each sample was assigned to the corresponding group based on the indicators. A first analysis involved the search of taxonomic indicators for all the set of samples including three groups of sites of low, medium and high impact of emerging contamination. It was possible to find indicators with a very high IndVal value for the three groups of samples. All indicators were based on the co-occurrence of three ASVs belonging to several of the most abundant bacteria phyla (Actinobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria). The bacterial indicators correctly assigned 100% and 93% of the samples to their corresponding group for streams and lagoons respectively. Then, a comparison between taxonomic and functional indicators using a subset of 41 samples was made, including two groups of samples: high and low-medium impact. Both the taxonomic and functional indicators showed high IndVal values for the high and low impact groups, being the highest in the case of functional genes Table 1. The high impact group was perfectly predicted for both taxonomic and functional indicators. The low-medium impact group was perfectly predicted by the functional indicators and 85% of the samples were correctly assigned by the taxonomic indicators. In conclusion, widespread availability of NGS technology allows for deep characterization of microbial diversity, enabling the use of robust ecological tools. Taking into account the high indval and prediction values, taxonomic and functional bacterial indicators appear as promissory candidates to evaluate for aquatic systems monitoring and conservation strategies.

The complete mitochondrial genome of Trematomus loennbergii (Perciformes, Nototheniidae)

The complete mitochondrial genome of Trematomus loennbergii was studied using NGS technology with PacBio platform. The mitochondrial genome size was 19,374bp and it had 13 protein-coding genes, 22 tRNAs and 2 rRNAs. There were 4 types of stop codons which were TAA, TAG, AGG and T(AA) but start codon type was only one (ATG). The contents of GC were 44.09% and AT contents were 55.91%. To conduct phylogenetic analysis, 12 species in 3 families were used. The result suggested that T. loennbergii was close to Pagothenia borchgrevinki in Nototheniidae. This study would provide a fundamental data for molecular evolution of T. loennbergii.

Routine Molecular Screening of Patients with Advanced Non-SmallCell Lung Cancer in Circulating Cell-Free DNA at Diagnosis and During Progression Using OncoBEAMTM EGFR V2 and NGS Technologies

Background and ObjectivesThe use of ultra-sensitive diagnostic tests to detect clinically actionable somatic alterations within the gene encoding the epidermal growth factor receptor (EGFR) within circulating cell-free DNA is an important first step in determining the eligibility of patients with non-small cell lung cancer to receive tyrosine kinase inhibitors.MethodsWe present the clinical validation (accuracy, sensitivity, and specificity) of a highly sensitive OncoBEAMTM EGFR V2 test, which we compare to a custom next-generation sequencing assay, for the treatment of patients with non-small cell lung cancer with EGFR tyrosine kinase inhibitor therapies. The OncoBEAMTM digital-polymerase chain reaction method detects 36 different EGFR alterations in circulating cell-free DNA, whereas the next-generation sequencing assay covers major solid tumor oncodrivers. Of the 540 samples analyzed with the OncoBEAMTM EGFR V2 test, 42.4% of patients had undergone molecular testing at diagnosis (N = 229/540) and 57.7% of patients during disease progression (N = 311/540).ResultsThe sensitivity and specificity were measured for this BEAMing assay. The number of mutant beads and mutant allelic fraction were measured for each EGFR alteration and the level of detection was established at 0.1% for a median of 2861 genome equivalent (GE) in each reaction using HD780 horizon control DNA, as well as by an internal quality reference standard. Approximately 10%, 27%, and 63% of the 540 samples contained < 1500 GE, a range of 1500–3000 GE, and > 3000 GE, which corresponded to a maximal assay sensitivity of 2.0%, 0.5–0.1%, and 0.1–0.05% mutant allelic fraction, respectively. In a routine hospital setting, 11.4% of non-small cell lung cancer tumors were positive at diagnosis for EGFR alterations, while 43.7% samples harbored EGFR mutations at progression, among which 40.3% expressed EGFR resistance mutations after first-line tyrosine kinase inhibitor treatment with first- and second-generation drugs.ConclusionsThe OncoBEAMTM EGFR V2 is a sensitive, robust, and accurate assay that delivers reproducible results. Next-generation sequencing and BEAMing technologies act complementarily in the routine molecular screening. We show that using a next-generation sequencing assay, despite its lower sensitivity, enables the identification of rare EGFR alterations or resistance mechanisms (mutation, deletion, insertion, and copy number variation) to orient first- and second-line treatments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00515-9.

PVT1: A long non-coding RNA recurrently involved in neoplasia-associated fusion transcripts.

NGS technologies and bioinformatics tools allow the rapid identification of chimeric transcripts in cancer. More than 40,000 fusions are so far reported in the literature; however, for most of them, the role in oncogenesis is still not fully understood. This is the case for fusions involving the long non-coding RNA (lncRNA) Plasmacytoma variant translocation 1 (PVT1) (8q24.21). This lncRNA displays oncogenic functions in several cancer types interacting with microRNAs and proteins, but the role of PVT1 fusion transcripts is more obscure. These chimeras have been identified in both hematological malignancies and solid tumors, mainly arising from rearrangements and/or amplification of the 8q24 chromosomal region. In this review, we detail the full spectrum of PVT1 fusions in cancer, summarizing current knowledge about their genesis, function, and role as biomarkers.

Abstract PS7-54: Racial differences in age-related DNA methylation changes in normal breast tissue

Abstract Age is a well-established risk factor for breast cancer development. While age-related epigenetic changes are believed to contribute to overall breast cancer risk, the underlying molecular mechanisms are poorly understood. In addition, racial disparity in breast cancer is well recognized. Compared to European American (EA) women, African American (AA) women are more often diagnosed with breast cancer at younger ages, have a more advanced or aggressive disease, and have poorer outcomes. We hypothesize that age-related DNA methylation (DNAm) may contribute to breast cancer risk differently for distinct racial groups. We investigated epigenome-wide age-related DNAm in normal breast tissue from 178 EA and 272 AA women separately using the Illumina TruSeq Methyl Capture EPIC library and NGS technology. We identified 3,944 and 506 CpG loci that were significantly associated with chronological age, of which 3,534 and 504 CpGs were increasingly hypermethylated with older ages and 410 and 2 loci were increasingly hypomethylated with older ages, in EA and AA women, respectively. Despite the different number, all 506 loci that were significantly associated with age in AA women were also significant for EA women. The low number age-related DNAm loci identified in AA women may reflect a more complex interplay of factors on epigenetic mechanisms in this racial group. Pathway analyses suggested these age-related loci are enriched in biological processes including cell communication, signaling, proliferation, and adhesion. We further examined 181 CpG loci that were previously reported to be associated with age in normal breast tissue but found that only six loci were replicated in our dataset. Our results suggested age-related biological pathways that potentially implicated in breast cancer development and a more complex epigenetic mechanisms in AA women. Citation Format: Chunyan He, James Castle, Nan Lin, Yunlong Liu, Chi Wang. Racial differences in age-related DNA methylation changes in normal breast tissue [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-54.

Recent advancement of NGS technologies to detect active transposable elements in plants.

Unlike peoples' belief that transposable elements (TEs) are "junk DNAs" or "genomic parasites", TEs are essential genomic elements that bring about genetic diversity and enable evolution of a species. In fact, transposons are major constituent of chromosome in crop genomes, particularly in major cereal crops, the primary type of which is long terminal repeat (LTR) retrotransposon. Since TE mobilization can be controlled by specific environmental stimulation and as the result can generate novel genetic variations, it has been suggested that controlled mobilization of TEs can be a plausible method for crop breeding. To achieve this goal, series of sequencing techniques have been recently established to identify TEs that are active in mobility. These methods target and detect extrachromosomal DNAs (ecDNAs), which are final products of integration. The newly identified TEs by these methods exhibit strong transpositional activity which can generate novel genetic diversity and provide useful breeding resources. In this mini review, we summarize and introduce ALE-seq, mobilome-seq, and VLP DNA-seq techniques employed to detect active TEs in plants.

Selection of reference genes for normalization of microRNA expression in sugarcane stalks during its interaction with Colletotrichum falcatum.

The microRNAs role in various cellular and metabolic functions is gaining more limelight in line with second-generation NGS technology. For the validation of candidate miRNA genes, the quantitative real-time PCR is the widely trusted and efficient method to follow. Sugarcane miRNAs are less explored in sugarcane defense response during their interaction with Colletotrichum falcatum inciting red rot. Further, for RT-qPCR experiments involving sugarcane miRNA expression studies, a stable internal reference gene is required. Hence, we have taken a study involving 20 candidate genes to identify stable expressing reference genes using NormFinder, geNorm, BestKeeper, and deltaCt statistical algorithms. The candidate reference genes included miRNAs and protein-coding genes. The results indicated that there is a variation in ranking among the algorithms. We found miR1862c as the stably expressed miRNA reference gene among the candidates and miR444b.2 along miR1862c formed the best reference gene pair combination, which can be used in the experiments aiming to explore sugarcane miRNAs in the defense mechanism against C. falcatum. The stable miRNA reference gene was further validated with other lesser stable reference gene candidates to assess the effect of stable reference genes during normalization. The present study evaluating the sugarcane miRNAs as reference genes for normalizing RT-qPCR expression data involving miRNAs during sugarcane ×C. falcatum interaction is the first of its kind. Further, this systematic approach can be followed to assess the reference gene in various experimental conditions involving sugarcane miRNAs.

The Importance of Targeted Next-Generation Sequencing Usage in Cytogenetically Normal Myeloid Malignancies.

Advanced diagnostic methods give an advantage for the identification of abnormalities in myeloid malignancies. Various researchers have shown the potential importance of genetic tests before the disease’s onset and in remission. Large testing panels prevent false-negative results in myeloid malignancies. However, the critical question is how the results of conventional cytogenetic and molecular cytogenetic techniques can be merged with NGS technologies. In this paper, we drew an algorithm for the evaluation of myeloid malignancies. To evaluate genetic abnormalities, we performed cytogenetics, molecular cytogenetics, and NGS testing in myeloid malignancies. In this study, we analyzed 100 patients admitted to the Medical Genetics Laboratory with different myeloid malignancies. We highlighted the possible diagnostic algorithm for cytogenetically normal cases. We applied NGS 141 gene panel for cytogenetically normal patients, and we detected two or more pathogenic variations in 61 out of 100 patients (61%). NGS’s pathogenic variation detection rate varies in disease groups: they were present in 85% of A.M.L. and 23% of M.D.S. Here, we identified 24 novel variations out of total pathogenic variations in myeloid malignancies. A total of 18 novel variations were identified in A.M.L., and 6 novel variations were identified in M.D.S. Despite long turnaround times, conventional techniques are still a golden standard for myeloid malignancies but sometimes cryptic gene fusions or complex abnormalities cannot be easily identified by conventional techniques. In these conditions, advanced technologies like NGS are highly recommended.

Update on the intricate tango between tick microbiomes and tick-borne pathogens.

The recent development of high-throughput NGS technologies, (ie, next-generation sequencing) has highlighted the complexity of tick microbial communities-which include pathogens, symbionts, and commensals-and also their dynamic variability. Symbionts and commensals can confer crucial and diverse benefits to their hosts, playing nutritional roles or affecting fitness, development, nutrition, reproduction, defence against environmental stress and immunity. Nonpathogenic tick bacteria may also play a role in modifying tick-borne pathogen colonization and transmission, as relationships between microorganisms existing together in one environment can be competitive, exclusive, facilitating or absent, with many potential implications for both human and animal health. Consequently, ticks represent a compelling yet challenging system in which to investigate the composition and both the functional and ecological implications of tick bacterial communities, and thus merits greater attention. Ultimately, deciphering the relationships between microorganisms carried by ticks as well as symbiont-tick interactions will garner invaluable information, which may aid in some future arthropod-pest and vector-borne pathogen transmission control strategies. This review outlines recent research on tick microbiome composition and dynamics, highlights elements favouring the reciprocal influence of the tick microbiome and tick-borne agents and finally discusses how ticks and tick-borne diseases might potentially be controlled through tick microbiome manipulation in the future.

High-Throughput Metagenomics for Identification of Pathogens in the Clinical Settings.

The application of sequencing technology is shifting from research to clinical laboratories owing to rapid technological developments and substantially reduced costs. However, although thousands of microorganisms are known to infect humans, identification of the etiological agents for many diseases remains challenging as only a small proportion of pathogens are identifiable by the current diagnostic methods. These challenges are compounded by the emergence of new pathogens. Hence, metagenomic next‐generation sequencing (mNGS), an agnostic, unbiased, and comprehensive method for detection, and taxonomic characterization of microorganisms, has become an attractive strategy. Although many studies, and cases reports, have confirmed the success of mNGS in improving the diagnosis, treatment, and tracking of infectious diseases, several hurdles must still be overcome. It is, therefore, imperative that practitioners and clinicians understand both the benefits and limitations of mNGS when applying it to clinical practice. Interestingly, the emerging third‐generation sequencing technologies may partially offset the disadvantages of mNGS. In this review, mainly: a) the history of sequencing technology; b) various NGS technologies, common platforms, and workflows for clinical applications; c) the application of NGS in pathogen identification; d) the global expert consensus on NGS‐related methods in clinical applications; and e) challenges associated with diagnostic metagenomics are described.

Candidate gene association of gene expression data in sugarcane contrasting for sucrose content

Association mapping of gene expression data, generated from transcriptome and proteome studies, provides a means of understanding the functional significance and trait association potential of candidate genes. In this study, we applied candidate gene association mapping to validate sugarcane genes, using data from the starch and sucrose metabolism pathway, transcriptome, and proteome. We performed multiplex PCR targeted amplicon sequencing of 109 candidate genes, using NGS technology. A range of statistical models, both single-locus and multi-locus, were compared for minimization of false positives in association mapping of four sugar-related traits with different heritability. The Fixed and random model Circulating Probability Unification model effectively suppressed false positives for both low- and high-heritability traits. We identified favorable alleles of the candidate genes involved in signalling and transcriptional regulation. The results will support genetic improvement of sugarcane and may help clarify the genetic architecture of sugar-related traits.

Utility of the Mitochondrial Genome in Plant Taxonomic Studies.

Size, structure, and sequence content lability of plant mitochondrial genome (mtDNA) across species has sharply limited its use in taxonomic studies. Historically, mtDNA variation has been first investigated with RFLPs, while the development of universal primers then allowed studying sequence polymorphisms within short genomic regions (<3kb). The recent advent of NGS technologies now offers new opportunities by greatly facilitating the assembly of longer mtDNA regions, and even full mitogenomes. Phylogenetic works aiming at comparing signals from different genomic compartments (i.e., nucleus, chloroplast, and mitochondria) have been developed on a few plant lineages, and have been shown especially relevant in groups with contrasted inheritance of organelle genomes. This chapter first reviews the main characteristics of mtDNA and the application offered in taxonomic studies. It then presents tips for best sequencing protocol based on NGS data to be routinely used in mtDNA-based phylogenetic studies.

Ocena zmian profilu ekspresji genów kandydujących w podkładkach jabłoni o odmiennym stopniu tolerancji mrozowej

Celem przeprowadzonych badań była identyfikacja genów sprzężonych z cechą mrozoodporności podkładek jabłoni. Ocenę zmian w poziomie ekspresji wyizolowanych genów przeprowadzono metodami RNAseq i qRT-PCR, dla podkładek zróżnicowanych po względem stopnia tolerancji mrozowej: P 66 (tolerancyjna) i M.9 (wrażliwa).W wyniku przeprowadzonych odczytów sekwencji RNA (sekwencjonowanie de novo w systemie Illumina Solid) dla w/w podkładek zidentyfikowano około 167 milionów odczytów unikatowych sekwencji, z których do wstępnych badań weryfikacyjnych wytypowano 15 o zróżnicowanym profilu ekspresji. Sekwencje poddano adnotacji funkcjonalnej. Wytypowane geny kodują: białka strukturalne i integralne błon komórkowych i wakuoli komórkowych, czynników transkrypcyjnych, białek regulujących transport międzykomórkowy i wewnątrzkomórkowy, białek hydrolizujących wiązania C-O i C-N oraz białek wiążących makro- i mikroelementy. Celem weryfikacji typu regulacji sekwencji transkryptomu uzyskanych z sekwencjonowania nowej generacji (NGS), dla tych samych prób przeprowadzono ilościową analizę transkryptu genów (qRT-PCR). Spośród badanych genów, trzy reprezentowały identyczny typ regulacji w badanych układach eksperymentalnych RNA-seq i qRT-PCR. Wytypowane geny stanowią potencjalne sekwencje kandydujące do sporządzenia markerów funkcjonalnych, umożliwiających wczesną selekcję podkładek jabłoni tolerancyjnych na mróz.