Refining the pneumococcal competence regulon by RNA-sequencing

Refining the Pneumococcal Competence Regulon by RNA Sequencing

Journal of Bacteriology ◽

10.1128/jb.00780-18 ◽

2019 ◽

Vol 201 (13) ◽

Cited By ~ 11

Author(s):

Jelle Slager ◽

Rieza Aprianto ◽

Jan-Willem Veening

Keyword(s):

Antibiotic Resistance ◽

Streptococcus Pneumoniae ◽

Rna Sequencing ◽

Genome Annotation ◽

Stress Factors ◽

Microarray Technology ◽

Human Pathogen ◽

Exogenous Dna ◽

Content Type ◽

Opportunistic Human Pathogen

ABSTRACTCompetence for genetic transformation allows the opportunistic human pathogenStreptococcus pneumoniaeto take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniaeis an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

Transcriptomic Profiling of Skeletal Muscle Reveals Candidate Genes Influencing Muscle Growth and Associated Lipid Composition in Portuguese Local Pig Breeds

Animals ◽

10.3390/ani11051423 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1423

Author(s):

André Albuquerque ◽

Cristina Óvilo ◽

Yolanda Núñez ◽

Rita Benítez ◽

Adrián López-Garcia ◽

...

Keyword(s):

Candidate Genes ◽

Muscle Growth ◽

Rna Seq ◽

Next Generation Sequencing Technology ◽

Pig Breeds ◽

Nfkb Activation ◽

Main Gene ◽

Slow Type ◽

Longissimus Lumborum ◽

Generation Sequencing

Gene expression is one of the main factors to influence meat quality by modulating fatty acid metabolism, composition, and deposition rates in muscle tissue. This study aimed to explore the transcriptomics of the Longissimus lumborum muscle in two local pig breeds with distinct genetic background using next-generation sequencing technology and Real-Time qPCR. RNA-seq yielded 49 differentially expressed genes between breeds, 34 overexpressed in the Alentejano (AL) and 15 in the Bísaro (BI) breed. Specific slow type myosin heavy chain components were associated with AL (MYH7) and BI (MYH3) pigs, while an overexpression of MAP3K14 in AL may be associated with their lower loin proportion, induced insulin resistance, and increased inflammatory response via NFkB activation. Overexpression of RUFY1 in AL pigs may explain the higher intramuscular (IMF) content via higher GLUT4 recruitment and consequently higher glucose uptake that can be stored as fat. Several candidate genes for lipid metabolism, excluded in the RNA-seq analysis due to low counts, such as ACLY, ADIPOQ, ELOVL6, LEP and ME1 were identified by qPCR as main gene factors defining the processes that influence meat composition and quality. These results agree with the fatter profile of the AL pig breed and adiponectin resistance can be postulated as responsible for the overexpression of MAP3K14′s coding product NIK, failing to restore insulin sensitivity.

Different miRNA Profiles in Plasma Derived Small and Large Extracellular Vesicles from Patients with Neurodegenerative Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22052737 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2737

Author(s):

Daisy Sproviero ◽

Stella Gagliardi ◽

Susanna Zucca ◽

Maddalena Arigoni ◽

Marta Giannini ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Extracellular Vesicles ◽

Small Rnas ◽

Biomarker Discovery ◽

Toll Like Receptor ◽

Neurotrophin Signaling ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Lateral Sclerosis ◽

Glycosphingolipid Biosynthesis

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.

A mutation in LacDWARF1 results in a GA-deficient dwarf phenotype in sponge gourd (Luffa acutangula)

Theoretical and Applied Genetics ◽

10.1007/s00122-021-03938-4 ◽

2021 ◽

Author(s):

Gangjun Zhao ◽

Caixia Luo ◽

Jianning Luo ◽

Junxing Li ◽

Hao Gong ◽

...

Keyword(s):

Gene Annotation ◽

Recessive Gene ◽

Genomic Region ◽

Dwarf Mutant ◽

Rna Seq ◽

Dwarf Phenotype ◽

Sponge Gourd ◽

Response To Stress ◽

Luffa Acutangula ◽

Generation Sequencing

Abstract Key message A dwarfism gene LacDWARF1 was mapped by combined BSA-Seq and comparative genomics analyses to a 65.4 kb physical genomic region on chromosome 05. Abstract Dwarf architecture is one of the most important traits utilized in Cucurbitaceae breeding because it saves labor and increases the harvest index. To our knowledge, there has been no prior research about dwarfism in the sponge gourd. This study reports the first dwarf mutant WJ209 with a decrease in cell size and internodes. A genetic analysis revealed that the mutant phenotype was controlled by a single recessive gene, which is designated Lacdwarf1 (Lacd1). Combined with bulked segregate analysis and next-generation sequencing, we quickly mapped a 65.4 kb region on chromosome 5 using F2 segregation population with InDel and SNP polymorphism markers. Gene annotation revealed that Lac05g019500 encodes a gibberellin 3β-hydroxylase (GA3ox) that functions as the most likely candidate gene for Lacd1. DNA sequence analysis showed that there is an approximately 4 kb insertion in the first intron of Lac05g019500 in WJ209. Lac05g019500 is transcribed incorrectly in the dwarf mutant owing to the presence of the insertion. Moreover, the bioactive GAs decreased significantly in WJ209, and the dwarf phenotype could be restored by exogenous GA3 treatment, indicating that WJ209 is a GA-deficient mutant. All these results support the conclusion that Lac05g019500 is the Lacd1 gene. In addition, RNA-Seq revealed that many genes, including those related to plant hormones, cellular process, cell wall, membrane and response to stress, were significantly altered in WJ209 compared with the wild type. This study will aid in the use of molecular marker-assisted breeding in the dwarf sponge gourd.

Erratum: Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

Nature Biotechnology ◽

10.1038/nbt1114-1166a ◽

2014 ◽

Vol 32 (11) ◽

pp. 1166-1166 ◽

Cited By ~ 3

Author(s):

Sheng Li ◽

Scott W Tighe ◽

Charles M Nicolet ◽

Deborah Grove ◽

Shawn Levy ◽

...

Keyword(s):

Next Generation Sequencing ◽

Transcriptome Profiling ◽

Rna Seq ◽

Next Generation ◽

Generation Sequencing

Next-generation sequencing reveals two populations of damage-induced small RNAs at endogenous DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gky1107 ◽

2018 ◽

Vol 46 (22) ◽

pp. 11869-11882 ◽

Cited By ~ 25

Author(s):

Franziska Bonath ◽

Judit Domingo-Prim ◽

Marcel Tarbier ◽

Marc R Friedländer ◽

Neus Visa

Keyword(s):

Next Generation Sequencing ◽

Small Rnas ◽

Double Strand Breaks ◽

Next Generation ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Two Populations ◽

Generation Sequencing

Transcriptome-wide m6A detection with DART-Seq

10.21203/rs.2.12189/v1 ◽

2019 ◽

Author(s):

Kate D. Meyer

Keyword(s):

Gene Expression Regulation ◽

Preparation Method ◽

Current Knowledge ◽

Cross Reactivity ◽

High Variability ◽

Library Preparation ◽

Rna Seq ◽

Generation Sequencing ◽

Library Preparation Method

Abstract m6A is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about m6A has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global m6A mapping studies. These methods rely on m6A antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify m6A-containing transcripts1,2. While these methods enabled the first identification of m6A sites transcriptome-wide and have dramatically improved our ability to study m6A, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and m6A antibody cross-reactivity with other modifications. Here, we describe DART-Seq (deamination adjacent to RNA modification targets), an antibody-free method for global m6A detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the m6A-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with in vitro assays, and subsequent deamination of m6A-adjacent cytidines is then detected by RNA sequencing to identify m6A sites. DART-Seq can successfully map m6A sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

BioSeqZip: a collapser of NGS redundant reads for the optimization of sequence analysis

Bioinformatics ◽

10.1093/bioinformatics/btaa051 ◽

2020 ◽

Vol 36 (9) ◽

pp. 2705-2711 ◽

Cited By ~ 2

Author(s):

Gianvito Urgese ◽

Emanuele Parisi ◽

Orazio Scicolone ◽

Santa Di Cataldo ◽

Elisa Ficarra

Keyword(s):

Sequence Analysis ◽

Supplementary Information ◽

Sorting Algorithm ◽

Rna Seq ◽

Compact Sets ◽

Analysis Pipeline ◽

Alignment Algorithms ◽

External Sorting ◽

Computational Resources ◽

Generation Sequencing

Abstract Motivation High-throughput next-generation sequencing can generate huge sequence files, whose analysis requires alignment algorithms that are typically very demanding in terms of memory and computational resources. This is a significant issue, especially for machines with limited hardware capabilities. As the redundancy of the sequences typically increases with coverage, collapsing such files into compact sets of non-redundant reads has the 2-fold advantage of reducing file size and speeding-up the alignment, avoiding to map the same sequence multiple times. Method BioSeqZip generates compact and sorted lists of alignment-ready non-redundant sequences, keeping track of their occurrences in the raw files as well as of their quality score information. By exploiting a memory-constrained external sorting algorithm, it can be executed on either single- or multi-sample datasets even on computers with medium computational capabilities. On request, it can even re-expand the compacted files to their original state. Results Our extensive experiments on RNA-Seq data show that BioSeqZip considerably brings down the computational costs of a standard sequence analysis pipeline, with particular benefits for the alignment procedures that typically have the highest requirements in terms of memory and execution time. In our tests, BioSeqZip was able to compact 2.7 billion of reads into 963 million of unique tags reducing the size of sequence files up to 70% and speeding-up the alignment by 50% at least. Availability and implementation BioSeqZip is available at https://github.com/bioinformatics-polito/BioSeqZip. Supplementary information Supplementary data are available at Bioinformatics online.

Differential Expression Profiles of the Transcriptome and miRNA Interactome in Synovial Fibroblasts of Rheumatoid Arthritis Revealed by Next Generation Sequencing

Diagnostics ◽

10.3390/diagnostics9030098 ◽

2019 ◽

Vol 9 (3) ◽

pp. 98

Author(s):

Chia-Chun Tseng ◽

Ling-Yu Wu ◽

Wen-Chan Tsai ◽

Tsan-Teng Ou ◽

Cheng-Chin Wu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Differential Expression ◽

Regulatory Network ◽

Functional Annotation ◽

Interaction Analysis ◽

Expression Profiles ◽

Synovial Fibroblasts ◽

Rna Seq ◽

Network Mapping ◽

Generation Sequencing

Using next-generation sequencing to decipher the molecular mechanisms underlying aberrant rheumatoid arthritis synovial fibroblasts (RASF) activation, we performed transcriptome-wide RNA-seq and small RNA-seq on synovial fibroblasts from rheumatoid arthritis (RA) subject and normal donor. Differential expression of mRNA and miRNA was integrated with interaction analysis, functional annotation, regulatory network mapping and experimentally verified miRNA–target interaction data, further validated with microarray expression profiles. In this study, 3049 upregulated mRNA and 3552 downregulated mRNA, together with 50 upregulated miRNA and 35 downregulated miRNA in RASF were identified. Interaction analysis highlighted contribution of miRNA to altered transcriptome. Functional annotation revealed metabolic deregulation and oncogenic signatures of RASF. Regulatory network mapping identified downregulated FOXO1 as master transcription factor resulting in altered transcriptome of RASF. Differential expression in three miRNA and corresponding targets (hsa-miR-31-5p:WASF3, hsa-miR-132-3p:RB1, hsa-miR-29c-3p:COL1A1) were also validated. The interactions of these three miRNA–target genes were experimentally validated with past literature. Our transcriptomic and miRNA interactomic investigation identified gene signatures associated with RASF and revealed the involvement of transcription factors and miRNA in an altered transcriptome. These findings help facilitate our understanding of RA with the hope of serving as a springboard for further discoveries relating to the disease.

Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events

Blood ◽

10.1182/blood-2010-12-324517 ◽

2011 ◽

Vol 118 (7) ◽

pp. 1903-1911 ◽

Cited By ~ 83

Author(s):

Luca Cecchetti ◽

Neal D. Tolley ◽

Noemi Michetti ◽

Loredana Bury ◽

Andrew S. Weyrich ◽

...

Keyword(s):

Next Generation Sequencing ◽

Matrix Metalloproteinases ◽

Mrna Expression ◽

Vascular Remodeling ◽

Differentially Express ◽

Expression Patterns ◽

Critical Role ◽

Rna Seq ◽

Next Generation ◽

Generation Sequencing

Abstract Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.