Current Next-generation Sequencing Research Articles

Therapeutic implications of etiology-specific diagnosis of early-onset developmental and epileptic encephalopathies (EO-DEEs): A nationwide Turkish cohort study

ObjectiveTo evaluate the etiology-specific diagnosis of early-onset developmental epileptic encephalopathies (EO-DEEs) in a nationwide Turkish cohort to determine the implications for therapeutic management. MethodsThe cohort comprised 1450 patients who underwent EO-DEE. The utility of genetic testing was assessed with respect to the initial phases of next generation sequencing (NGS) (2005–2013) and the current NGS era (2014–2022). A predefined four-stepwise diagnostic model was evaluated using cost-effectiveness analysis. The diagnostic and potential therapeutic yields of the genetic tests were subsequently determined. ResultsGene-related EO-DEEs were identified in 48.3% (n=701) of the cohort: non-structural genetic (62.6%), metabolic genetic (15.1%), and structural genetic (14.1%). The most common nonstructural genetic variants were SCN1A (n=132, 18.8%), CDKL5 (n=30, 4.2%), STXBP1 (n=21, 2.9%), KCNQ2 (n=21, 2.9%), and PCDH19 (n=17, 2.4%). The rate of ultra-rare variants (< 0.5%) was higher in the NGS era (52%) than that in the initial phase (36%). The potential therapeutic yields with precision therapy and antiseizure drug modification were defined in 34.5% and 56.2% in genetic-EO-DEEs, respectively. The diagnostic model provided an etiology-specific diagnosis at a rate of 78.7%: structural (nongenetic) (31.4%), genetic (38.5%), metabolic (6.1%), and immune-infectious (2.8%). Based on a cost-effectiveness analysis, the presented diagnostic model indicated the early implementation of whole-exome sequencing for EO-DEEs. SignificanceIn the present cohort, the higher rate (48.3%) of gene-related EO-DEE diagnoses in the NGS era provides a potential therapeutic management plan for more patients.

Reproducible and high sample throughput isomiR next-generation sequencing for cancer diagnosis.

e15013 Background: Next-generation sequencing (NGS) can produce up to 6 Tb of data per run with single-nucleotide accuracy, making it ideal for quantifying isomiRs, which encompass both canonical miRNAs and their variants, for clinical applications. However, NGS has poor reproducibility and low sample throughput in quantifying circulating isomiRs due to significant technical variations and the limitations of the multiplex strategy, as evidenced by the fact that no isomiR NGS technique has been successfully used to diagnose cancer. Methods: To address these challenges, a library construction method including a dual unique-dual-index (DUDI) technology was developed. DUDI uses a pair of Inner UDI (IUDI) and outer UDI (OUDI) to label a sample. Twelve independent batches of isomiR NGS were carried out, including three repeated batches. Each batch included 100 gastric cancer and 100 control plasma samples. Batch effect, correlation coefficient (R), and principal component (PCA) analyses were used to evaluate technical reproducibility. Machine learning binary classification was used to assess biological reproducibility, with each pair of batch data serving interchangeably as both training and testing data. Results: In this multicenter study, over 700G of isomiR data were generated from 402 gastric cancer and 498 control samples, with a maximum error rate of 1 in 7 million isomiRs being assigned to wrong samples. The PCA plot indicates high technical reproducibility across the three repeated batches, shown by the extensive intermingling of data points from each batch and the lack of distinct batch-wise clustering. This observation is reinforced by that the R value for each of 239 isomiRs between the repeated batches are close to 1. While the mutual machine learning validations between the repeated batches yielded ~95% accuracy, indicating high biological reproducibility. The accuracies of the validations between the different batches of different samples range from 70% to 82%. The lower accuracy is as expected, given the high genetic heterogeneity of cancer and the small sample size. Furthermore, the IsomiR differentiated expression profiles from the current NGS study closely match those from prior qPCR studies. Conclusions: The DUDI library construction method can produce reproducible high sample throughput NGS data, yet it is cost-effective and straightforward. The maximum number of samples that can be multiplexed in an NGS project is almost one million, i.e., 976 * 976, as IUDI and OUDI can be any of the 976 designed DUDIs. This number far exceeds the high sample throughput requirements of any NGS application. While the capability to distinguish true biological variations of IsomiRs from technical noise, demonstrated by the high technical and biological reproducibility and concordance with the qPCR data, enables the development of robust machine learning algorithms for cancer diagnostics.

APPLICATION OF THE NEXT GENERATION SEQUENCING IN BIOLOGY AND MEDICINE

Next-Generation Sequencing (NGS), also known as high-throughput sequencing, refers to a set of modern DNA sequencing technologies that have revolutionized the field of genomics. Advantages of NGS techniques involving high speed (parallel sequencing is faster than traditional methods, allowing researchers to obtain results more quickly), cost-effectiveness (ability to sequence multiple fragments simultaneously reduces the cost per base compared to traditional sequencing), and scalability (platforms can be scaled to accommodate varying levels of throughput depending on experimental needs). NGS has significantly accelerated genomics research, enabling breakthroughs in fields such as personalized medicine, cancer genomics, and evolutionary biology. However, challenges such as data analysis complexity, error rates, and cost still exist and are areas of ongoing research and improvement within the field of sequencing technologies. Paper contains the brief explanation of the current NGS platforms and their features. NGS biomedical application is described with the main advantages and abilities of the analysed tools.

The impact of misclassification errors on the performance of biomarkers based on next-generation sequencing, a simulation study

ABSTRACT The development of next-generation sequencing (NGS) opens opportunities for new applications such as liquid biopsy, in which tumor mutation genotypes can be determined by sequencing circulating tumor DNA after blood draws. However, with highly diluted samples like those obtained with liquid biopsy, NGS invariably introduces a certain level of misclassification, even with improved technology. Recently, there has been a high demand to use mutation genotypes as biomarkers for predicting prognosis and treatment selection. Many methods have also been proposed to build classifiers based on multiple loci with machine learning algorithms as biomarkers. How the higher misclassification rate introduced by liquid biopsy will affect the performance of these biomarkers has not been thoroughly investigated. In this paper, we report the results from a simulation study focused on the clinical utility of biomarkers when misclassification is present due to the current technological limit of NGS in the liquid biopsy setting. The simulation covers a range of performance profiles for current NGS platforms with different machine learning algorithms and uses actual patient genotypes. Our results show that, at the high end of the performance spectrum, the misclassification introduced by NGS had very little effect on the clinical utility of the biomarker. However, in more challenging applications with lower accuracy, misclassification could have a notable effect on clinical utility. The pattern of this effect can be complex, especially for machine learning-based classifiers. Our results show that simulation can be an effective tool for assessing different scenarios of misclassification.

Toward Cytogenomics: Technical Assessment of Long-Read Nanopore Whole-Genome Sequencing for Detecting Large Chromosomal Alterations in Mantle Cell Lymphoma

The current advances and success of next-generation sequencing hold the potential for the transition of cancer cytogenetics toward comprehensive cytogenomics. However, the conventional use of short reads impedes the resolution of chromosomal aberrations. Thus, this study evaluated the detection and reproducibility of extensive copy number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies whole-genome sequencing compared with short-read Illumina sequencing. Using the mantle cell lymphoma cell line Granta-519, almost 99% copy-number reproducibility at the 100-kilobase resolution between replicates was demonstrated, with 98% concordance to Illumina. Collectively, the performance of copy number calling from 1.5 million to 7.5 million long reads was comparable to 1 billion Illumina-based reads (50× coverage). Expectedly, the long-read resolution of canonical translocation t(11;14)(q13;q32) was superior, with a sequence similarity of 89% to the already published CCND1/IGH junction (9× coverage), spanning up to 69 kilobases. The cytogenetic profile of Granta-519 was in general agreement with the literature and karyotype, although several differences remained unresolved. In conclusion, contemporary long-read sequencing is primed for future cytogenomics or sequencing-guided cytogenetics. The combined strength of long- and short-read sequencing is apparent, where the high-precision junctional mapping complements and splits paired-end reads. The potential is emphasized by the flexible single-sample genomic data acquisition of Oxford Nanopore Technologies with the high resolution of allelic imbalances using Illumina short-read sequencing.

Alternative Splicing, RNA Editing, and the Current Limits of Next Generation Sequencing.

The advent of next generation sequencing (NGS) has fostered a shift in basic analytic strategies of a gene expression analysis in diverse pathologies for the purposes of research, pharmacology, and personalized medicine. What was once highly focused research on individual signaling pathways or pathway members has, from the time of gene expression arrays, become a global analysis of gene expression that has aided in identifying novel pathway interactions, the discovery of new therapeutic targets, and the establishment of disease-associated profiles for assessing progression, stratification, or a therapeutic response. But there are significant caveats to this analysis that do not allow for the construction of the full picture. The lack of timely updates to publicly available databases and the "hit and miss" deposition of scientific data to these databases relegate a large amount of potentially important data to "garbage", begging the question, "how much are we really missing?" This brief perspective aims to highlight some of the limitations that RNA binding/modifying proteins and RNA processing impose on our current usage of NGS technologies as relating to cancer and how not fully appreciating the limitations of current NGS technology may negatively affect therapeutic strategies in the long run.

Current clinical practices and challenges in molecular testing: a GOAL Consortium Hematopathology Working Group report

AbstractWhile molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

Exome/Genome Sequencing in Undiagnosed Syndromes.

Exome sequencing (ES) and genome sequencing (GS) have radically transformed the diagnostic approach to undiagnosed rare/ultrarare Mendelian diseases. Next-generation sequencing (NGS), the technology integral for ES, GS, and most large (100+) gene panels, has enabled previously unimaginable diagnoses, changes in medical management, new treatments, and accurate reproductive risk assessments for patients, as well as new disease gene discoveries. Yet, challenges remain, as most individuals remain undiagnosed with current NGS. Improved NGS technology has resulted in long-read sequencing, which may resolve diagnoses in some patients who do not obtain a diagnosis with current short-read ES and GS, but its effectiveness is unclear, and it is expensive. Other challenges that persist include the resolution of variants of uncertain significance, the urgent need for patients with ultrarare disorders to have access to therapeutics, the need for equity in patient access to NGS-based testing, and the study of ethical concerns. However, the outlook for undiagnosed disease resolution is bright, due to continual advancements in the field.

Budget impact analysis of next-generation sequencing versus sequential single-gene testing in Japanese patients with advanced non-small-cell lung cancer

BackgroundIdentification of genomic alterations (e.g., EGFR, ALK, ROS1, BRAF, NTRK, and MET) is essential for initiating targeted therapy in patients with advanced non-small-cell lung cancer (aNSCLC). This study estimated the budget impact of using the sequential single-gene (SSG) test, which tests for each mutation one at a time, versus next-generation sequencing (NGS), which tests for all mutations at the same time, among newly diagnosed patients with aNSCLC from a Japanese healthcare payer’s perspective. MethodsA budget impact model (BIM) was used to determine the expected budget impact associated with NGS for newly diagnosed aNSCLC in Japan over a 3-year period. The BIM compared the total costs (biopsy, testing, and treatment) and average turnaround time of “future NGS” and “current NGS” versus SSG testing. ResultsThe adoption of current NGS over SSG testing had a budget impact of –0.24%, but adoption of future NGS over SSG testing had a budget impact of +4.33% across a 3-year time horizon on the Japanese budget for aNSCLC treatment. The adoption of current or future NGS over SSG testing would shorten the average turnaround time for testing. ConclusionsThe adoption of current NGS over SSG testing would slightly decrease the yearly costs. However, the adoption of future or current NGS over SSG testing would shorten the average turnaround time, enabling faster identification of genomic alterations and earlier initiation of treatment for aNSCLC patients in Japan.

Genome-Wide Analysis of Periodontal and Peri-implant Cells and Tissues.

-Omics analyses, including the systematic cataloging of messenger RNA and microRNA sequences or DNA methylation patterns in a cell population, organ, or tissue sample, are powerful means of generating comprehensive genome-level data sets on complex diseases. We have systematically assessed the transcriptome, microbiome, miRNome, and methylome of gingival and peri-implant tissues from human subjects and further studied the transcriptome of primary cells ex vivo, or in vitro after infection with periodontal pathogens.Our data offer new insight on the pathophysiology underlying periodontal and peri-implant diseases, a possible route to a better and earlier diagnosis of these highly prevalent chronic inflammatory diseases and thus, to a personalized and efficient treatment approach.Herein, we outline the laboratory steps required for the processing of periodontal cells and tissues for -omics analyses using current microarrays or next-generation sequencing technology.

DirectRMDB: a database of post-transcriptional RNA modifications unveiled from direct RNA sequencing technology.

With advanced technologies to map RNA modifications, our understanding of them has been revolutionized, and they are seen to be far more widespread and important than previously thought. Current next-generation sequencing (NGS)-based modification profiling methods are blind to RNA modifications and thus require selective chemical treatment or antibody immunoprecipitation methods for particular modification types. They also face the problem of short read length, isoform ambiguities, biases and artifacts. Direct RNA sequencing (DRS) technologies, commercialized by Oxford Nanopore Technologies (ONT), enable the direct interrogation of any given modification present in individual transcripts and promise to address the limitations of previous NGS-based methods. Here, we present the first ONT-based database of quantitative RNA modification profiles, DirectRMDB, which includes 16 types of modification and a total of 904,712 modification sites in 25 species identified from 39 independent studies. In addition to standard functions adopted by existing databases, such as gene annotations and post-transcriptional association analysis, we provide a fresh view of RNA modifications, which enables exploration of the epitranscriptome in an isoform-specific manner. The DirectRMDB database is freely available at: http://www.rnamd.org/directRMDB/.

High‐frequency k‐mer counting at low memory footprint

Genomics data analysis requires efficient tools to address the vast amount of data generated by current next-generation sequencing technologies. K-mer counting works face difficulties in balancing high memory overhead with statistical precision. We designed a high-frequency k-mer statistical computation based on the Space Saving algorithm and a novel hash table structure, which reduces the memory overhead by 46% while ensuring high computational efficiency.

SparkEC: speeding up alignment-based DNA error correction tools

BackgroundIn recent years, huge improvements have been made in the context of sequencing genomic data under what is called Next Generation Sequencing (NGS). However, the DNA reads generated by current NGS platforms are not free of errors, which can affect the quality of downstream analysis. Although error correction can be performed as a preprocessing step to overcome this issue, it usually requires long computational times to analyze those large datasets generated nowadays through NGS. Therefore, new software capable of scaling out on a cluster of nodes with high performance is of great importance.ResultsIn this paper, we present SparkEC, a parallel tool capable of fixing those errors produced during the sequencing process. For this purpose, the algorithms proposed by the CloudEC tool, which is already proved to perform accurate corrections, have been analyzed and optimized to improve their performance by relying on the Apache Spark framework together with the introduction of other enhancements such as the usage of memory-efficient data structures and the avoidance of any input preprocessing. The experimental results have shown significant improvements in the computational times of SparkEC when compared to CloudEC for all the representative datasets and scenarios under evaluation, providing an average and maximum speedups of 4.9times and 11.9times, respectively, over its counterpart.ConclusionAs error correction can take excessive computational time, SparkEC provides a scalable solution for correcting large datasets. Due to its distributed implementation, SparkEC speed can increase with respect to the number of nodes in a cluster. Furthermore, the software is freely available under GPLv3 license and is compatible with different operating systems (Linux, Windows and macOS).

Using gene panels in the diagnosis of neuromuscular disorders: A mini-review.

The diagnosis of inherited neuromuscular disorders is challenging due to their genetic and phenotypic variability. Traditionally, neurophysiology and histopathology were primarily used in the initial diagnostic approach to these conditions. Sanger sequencing for molecular diagnosis was less frequently utilized as its application was a time-consuming and cost-intensive process. The advent and accessibility of next-generation sequencing (NGS) has revolutionized the evaluation process of genetically heterogenous neuromuscular disorders. Current NGS diagnostic testing approaches include gene panels, whole exome sequencing (WES), and whole genome sequencing (WGS). Gene panels are often the most widely used, being more accessible due to availability and affordability. In this mini-review, we describe the benefits and risks of clinical genetic testing. We also discuss the utility, benefits, challenges, and limitations of using gene panels in the evaluation of neuromuscular disorders.

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling.

During the past decade, breakthroughs in sequencing technology have provided the basis for studies of the myriad ways in which microbial communities in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and quantitative profiling of the microbiota for use in diagnostic and therapeutic applications. However, the current next-generation sequencing approach for microbiome profiling is costly, requires laborious library preparation, and is challenging to scale up for routine diagnostics. Split, Amplify, and Melt analysis of BActeria-community (SAMBA), a novel multicolor digital melting polymerase chain reaction platform with unprecedented multiplexing capability is presented, and the capability to distinguish and quantify 16 bacteria species in mixtures is demonstrated. Subsequently, SAMBA is applied to measure the compositions of bacteria in the gut microbiome to identify microbial dysbiosis related to colorectal cancer. This rapid, low cost, and high-throughput approach will enable the implementation of microbiome diagnostics in clinical laboratories and routine medical practice.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

FC049: Nefecon® (Budesonide) is an Epigenetic Modifier in IGA Nephropathy

Abstract BACKGROUND AND AIMS In recent years, the dysregulated expression of microRNAs (miRs)—small molecules of noncoding RNA that epigenetically fine-tune post-transcriptional gene expression—has been increasingly implicated in the pathogenesis of IgA nephropathy (IgAN). Extracellular miRs, contained within extracellular vesicles such as exosomes and microvesicles, are thought to function as important intercellular and interorgan modes of communication in health and disease. In the current study, next-generation sequencing (NGS) and subsequent real-time quantitative polymerase chain reaction (RT-qPCR) validation were performed to identify serum miRs associated with a high risk of future kidney function decline in IgAN. We then went on to determine whether treatment with Nefecon® 16 mg/day for 9 months was capable of modifying the serum levels of miRs associated with high-risk IgAN. This study was supported by a grant from Calliditas Therapeutics AB. METHOD NGS was performed by Qiagen (Germany) on serum from patients with IgAN (10 at high risk and 10 at low risk of progression) or membranous nephropathy (MN, n=10), as well as healthy individuals (HS, n=10). Sequencing data were validated by RT-qPCR using TaqMan miR PCR assays in independent sets of serum samples (20 per cohort). Using the Total Exosome Isolation (from serum) reagent (Thermo Fisher Scientific, UK), extracellular exosomes were isolated from the serum of participants of the NEFIGAN trial from the placebo group (n=40) and Nefecon® 16 mg/day group (n=40) at the start of treatment (SOT) and end of treatment (EOT). RNA was isolated and levels of miRs-483-5p and -122-5p were measured by RT-qPCR. The NEFIGAN trial (NCT01738035) was a randomized, double-blind, placebo-controlled Phase 2b trial designed to assess the safety and efficacy of Nefecon® a novel targeted-release formulation of budesonide, designed to deliver the drug to the distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. RESULTS miRs-483-5p and -122-5p were significantly overexpressed in the serum of patients with IgAN at high risk of progression compared with those who had stable IgAN or HS, but not compared with patients with MN. Both miRs were also found to be significantly increased in extracellular exosomes in high-risk IgAN compared with low-risk IgAN, HS and MN. Interestingly, levels of exosomal miRs-483-5p and -122-5p did not correlate with total serum IgA, IgA1, galactose-deficient IgA1 or IgA–IgG immune complex levels, suggesting a novel mechanism of action. Treatment with 16 mg/day of Nefecon® led to a significant reduction in the exosomal levels of miR-122-5p, while no change in miR-483-5p levels was observed in the treated group. CONCLUSION These data reveal a dysregulation of systemic exosomal miR expression in IgAN, and demonstrate for the first time that it is possible to pharmacologically modify the miR content of circulating exosomes in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively downregulated exosomal miR-122-5p, while having no effect on exosomal miR-483-5p levels, arguing against a generalized ‘anti-miR’ effect of Nefecon®. Studies are ongoing to both precisely delineate the cellular source(s) of these exosomes and determine the downstream impact of these changes on glomerular and tubulointerstitial biology.

Optimization and Validation of Nanopore Based Sequencing Method for Molecular Testing of CNS Tumours

BackgroundThe World Health Organization (WHO) introduced molecular identifiers for the diagnosis and prognosis of CNS tumors including the mutational status of isocitrate dehydrogenase or IDHgenes in glial tumors. Currently used immunohistochemistry (IHC) is not capable of detecting the non‐canonical mutations, and sequencing is often required as a follow‐up. Current next‐generation sequencing (NGS) technologies used in tumor molecular marker detection introduce key challenges including high capital cost, complex infrastructure requirements, and long turnaround times. These challenges considerably limit the ability to perform NGS testing in many pathology laboratories. In this study, we aimed to use third generation nanopore sequencing technology to resolve these limitations. The Oxford Nanopore MinION, a pocket‐sized nanopore sequencing device, has minimal capital costs and infrastructural requirements, and shorter turnaround times. However, the nanopore technology has not been validated in clinical practice and has not been optimized on formalin‐fixed paraffin‐embedded (FFPE) tissue.MethodsDNA extraction of selective tumor areas was performed from the corresponding FFPE tissue blocks from a cohort of gliomas with confirmed IDH1and IDH2gene statuses (n=65). A PCR amplicon‐based approach was used to amplify hot spots of the IDH1and IDH2genes, starting with 30ng DNA material. The amplicon libraries were sequenced for 2 hours in multiplex on the MinION device and IDHSNPs were called with the Nanopolish software. ASIP Abstract Mashiat MimosaResults26 IDH mutant samples were identified: 21 IDH1R132H, 2 IDH1R132G, 2 IDH2R172G, and 1 IDH2D177H. All cases showed concordant IDHmutational status when compared to the reference methods (IHC or NGS) and both analytical sensitivity and specificity were 100%. Precision analysis of variant allele frequency (VAF) showed the coefficient of variation was less than 5% (both inter and intra runs), and the limit of detection for VAF was 3%. The range of read depth obtained was 882X to 43,000x with an average of 20,000x. This assay revealed a $50‐$100 material cost per sample, and the time taken from extracted nucleic acid to final result generation was 1‐2 business days.ConclusionThis project is the first to optimize and validate an approach to detect SNP mutations in FFPE samples using nanopore technology. It has demonstrated the feasibility and efficacy of the nanopore amplicon sequencing method in cancer FFPE tissue with excellent test performance characteristics, significantly shorter turnaround times at considerably lower costs and without any infrastructural needs. Thus, it can be used to circumvent challenges to current NGS testing platforms and can be the milestone that would make cancer NGS testing available for every laboratory.

Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry.

RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation.

Combination analysis of single-molecule long-read and Illumina sequencing provides insights into the anthocyanin accumulation mechanism in an ornamental grass, Pennisetum setaceum cv. Rubrum

Combination analysis of single-molecule long-read and Illumina sequencing provide full-length transcriptome information and shed new light on the anthocyanin accumulation mechanism of Pennisetum setaceum cv. 'Rubrum'. Pennisetum setaceum cv. 'Rubrum' is an ornamental grass with purple leaves widely used in landscaping. However, the current next-generation sequencing (NGS) transcriptome information of this species is not satisfactory due to the difficulties in obtaining full-length transcripts. Furthermore, the molecular mechanisms of anthocyanin accumulation in P. setaceum have not been thoroughly studied. In this study, we used PacBio full-length transcriptome sequencing (SMRT) combined with NGS technology to build and improve the transcriptomic datasets and reveal the molecular mechanism of anthocyanin accumulation in P. setaceum cv. 'Rubrum'. Therefore, 280,413 full-length non-chimeric reads sequences were obtained using the SMRT technology. We obtained 97,450 high-quality non-redundant transcripts and identified 5352 alternative splicing events. In addition, 93,066 open reading frames (ORFs), including 57,457 full ORFs and 2910 long non-coding RNA (lncRNAs) were screened out. Furthermore, 10,795 differentially expressed genes were identified using NGS. We also explored key genes, synthesis pathways, and detected lncRNA involved in anthocyanin accumulation, providing new insights into anthocyanin accumulation in P. setaceum cv. 'Rubrum'. To our best knowledge, we provided the full-length transcriptome information of P. setaceum cv. 'Rubrum' for the first time. The results of this study will provide baseline information for gene function studies and pave the way for future P. setaceum cv. 'Rubrum' breeding projects.