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32 results for “Insertion sequences”
Generation of transcriptional novelty by transposable element insertions in Arabidopsis, Genome Sequencing and eccDNA Data
<p><strong>Raw Illumina sequencing data from the Manuscript entitled "Generation of transcriptional novelty by transposable element insertions in Arabidopsis"</strong></p> <p><strong>A. Illumina genome sequencing reads of Arabidopsis control and hcLines that contain novel transposable element insertions.</strong></p> <p>To identify the genomic position of the new <em>ONSEN</em> insertions, the extracted DNA of the 11 selected lines (nine lines with new insertions and two control lines) was sent to BGI, Hong-Kong for Illumina paired-end 150 bp sequencing, aiming for a minimum of 20X sequencing coverage. Quality control of the raw reads was done using FastQC (Andrews S. (2010). FastQC: a quality control tool for high throughput sequence data. Available online at: <a href="http://www.bioinformatics.babraham.ac.uk/projects/fastqc">http://www.bioinformatics.babraham.ac.uk/projects/fastqc</a>) and trimming/clipping was done using Trimmomatic with parameters ILLUMINACLIP: TruSeq3:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 and MINLEN:36. Quality of the reads was deemed excellent and no further actions were taken.</p> <p>Samples identifications: genome_hcLineX with "_1" indicating the forward and "_2" the reverse reads.</p> <p><strong>B. Illumina eccDNA sequencing of Arabidopsis control and hcLines following stress treatments</strong></p> <p>Extrachromosomal circular DNA was prepared and sequenced as follows: twenty plants from each petri dish were pooled separately and DNA was extracted using the CTAB method (<a href="https://dx.doi.org/10.17504/protocols.io.quidwue">dx.doi.org/10.17504/protocols.io.quidwue</a>). Following the mobilome-seq method described in (Lanciano et al., 2017), for all samples, we digested linear DNA from 2 µg of total DNA for 17 hours at 37<sup>o</sup>C using 10 U of PlasmidSafe (<em>LubioScience cat# E3101K</em>), followed by enzyme denaturation (30 mins at 70<sup>o</sup>C). Digested DNA was precipitated with isopropanol supplemented with 1 µg of GlycoBlue coprecipitant (<em>Fisher Scientific cat# 10391565</em>). Circular DNA was then amplified through rolling circle amplification (RCA) with the Illustra TempliPhi kit (<em>GE Healthcare cat# 25-6400-10</em>), following the manufacturer recommendation and leaving the reaction for 16h at 30<sup>o</sup>C. DNA was once again precipitated with isopropanol and sent for Illumina paired end 150 bp sequencing at BGI, Hong Kong. </p> <p>Samples identification: </p> <p>eccDNA_A.thaliana_ctrl: control reads</p> <p>eccDNA_A.thaliana_HS: heat stressed plants reads</p> <p>eccDNA_A.thaliana_AZ_HS: reads of alpha-amanitin, zebularine and heat-stressed plants</p> <p>"R1" indicates forward and "R2" reverse reads.</p> <p> </p>
Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteremia.
<p>Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in dissemination and modification of the antibiotic resistant phenotype. To that end, we leveraged fully resolved <em>Enterococcus faecium</em> and <em>Enterococcus faecalis</em> genomes of isolates collected over five days from an inpatient with prolonged bacteremia. Isolates from both species harbored similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of <em>E. faecium</em> isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in <em>E. faecalis</em> isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in <em>liaSR</em> genes and a putative composite transposon in<em> E. faecium</em>, offering a new paradigm for the study of daptomycin resistance and novel insights into the dissemination of daptomycin resistance. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.</p>
Sample datasets for Transposon insertion sequencing analysis tutorial
<p>The dataset contains five files:</p> <ol> <li>Tnseq-Tutorial-reads.fastqsanger.gz - A subset of TnSeq reads published in `Santiago, M., Matano, L. M., Moussa, S. H., Gilmore, M. S., Walker, S., & Meredith, T. C. (2015). A new platform for ultra-high density Staphylococcus aureus transposon libraries. <em>BMC Genomics</em>, <em>16</em>(1), 1–18. http://doi.org/10.1186/s12864-015-1361-3`</li> <li>condition_barcodes.fasta - Set of barcodes to separate reads from different experimental conditions</li> <li>construct_barcodes.fasta - Set of barcodes to separate reads from different transposon constructs</li> <li>staph_aur.fasta : Genome file for <em>Staphylococcus aureus </em></li> <li>staph_aur.fasta : Annotation file for <em>Staphylococcus aureus </em></li> </ol>
Generation of transcriptional novelty by transposable element insertions in Arabidopsis, RNAseq Control Condition Sequencing Data
<p><strong>Arabidopsis stranded 150 bp paired end RNA sequencing data (Illumina) of plants that were grown under control conditions for the manuscript "Generation of transcriptional novelty by transposable element insertions in Arabidopsis"</strong></p> <p><strong><strong>Plant growth conditions</strong></strong></p> <p>Sequenced F4 seeds were sterilized for 10 minutes in 10% bleach, rinsed, and stratified at 4°C for four days in the dark before being sown on 0.5x Murashige & Skoog media (Du<em>schefa cat# M0222</em>) and transferred to growth chambers under long day conditions (16h of light at 24°C followed by 8h of darkness at 21°C; 20 seeds per plate, 6 replicate plates). Ten days after sowing, plants were subjected to 6°C for 24 hours and control plants were returned to normal long day growing conditions for 24 hours before harvesting (3 replicate plates per condition).</p> <p><strong><strong>RNA extraction and sequencing</strong></strong></p> <p>Seedlings were harvested and RNA extractions were done on pools of 5 plants. RNA extractions were performed for 3 biological replicate samples for each line in each condition (n=96) using the Macherey-Nagel NucleoSpin RNA kit (cat# 740955.50). Samples were sent to Novogene for Illumina 150bp paired-end sequencing using a stranded poly-A library.</p> <p><strong>RNAseq sample descriptions of the plants grown under control conditions</strong></p> <p>wt_control: wild-type plants.</p> <p>wtHS_control: wild-type plants that have been submitted to heat stress in a previous generation.</p> <p>wtAZ_control: wild-type plants that have been submitted to epigenetic drug treatments (alpha-amanitin and zebularine) in a previous generation.</p> <p>htLine#: plants carrying additional <em>ONSEN</em> transposable element insertions.</p> <p>Files description: Forward and reverse strand RNA seq data are combined in one file. The numbering at the end ("_1") denominates the biological replicate number.</p>
ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing applied to identify small and large translated ORFs
<p>ProTInSeq is a novel -omics technique designed to characterize proteomes by using DNA ultra-deep sequencing. The technique is based on transposons engineered to have a positive or negative protein selection marker expressed when the transposon is inserted in-frame into a protein-coding gene. In the genome-reduced bacterium Mycoplasma pneumoniae, ProTInSeq identifies 80% of known expressed proteins, as well as 5 new open reading frames (ORFs; >100 amino acids); and 153 novel small ORF-encoded proteins (SEPs; ≤100 aa) that represent up to 18% of this bacterium’s proteome. ProTInSeq can be used to detect translational noise, for protein quantification and to provide insight into functional protein aspects such as relative half-life, stability, and membrane topology. Herein, we describe a methodology that can be easily implemented in any living system and allows the deep understanding of proteomes and more importantly the identification of small proteins by DNA ultra-sequencing.</p> <p>We include the following files:</p> <p>- processed_inscalling.zip: output obtain after running FASTQINS transposon calling tool over the datasets found at <a href="https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-10380?key=5f54209d-ce59-490a-9bcd-7084e9c619ee">https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-10380?key=5f54209d-ce59-490a-9bcd-7084e9c619ee</a>. This include every genome position in <em>M. pneumoniae</em>, the number of times an insertion has been mapped to that position and the total read count value.</p> <p>- separated_library_metrics.zip: insertion and read count processed from processed_inscalling files associated to every ORF and intergenic region in <em>M. penumoniae. </em>Columns include frame measured (0 - whole gene, 1 - in-frame, 2 and 3 for following positions) and metric. Metrics account for number insertions in-frame (<em>I</em>), read count (<em>R</em>), linear density from non-coding regions used in the Poisson evaluation (<em>rNC</em>), probability measured (<em>sfNC</em>) and a binary for prediction (<em>pred</em>; 0 - no significant, 1 - significant).</p> <p>- allmetrics.xlsx: merged table with the combination of results from separated_library_metrics.zip tab-delimited files.</p> <p>- selective_metrics_allannotations.xlsx: this table includes all the available information about the 30,112 sequences that could encode for a coding sequence in <em>M. pneumoniae</em>. For each identifier (column B), we include coordinates information and nucleotide and amino acid length information (columns C-H). Column I includes the gene name when the entry is found annotated in <em>M. pneumoniae</em>. Localization and function are described in columns J and K. Column L includes the operon number in which the annotation would be expressed. We also included transcription-related information average expression (column M; as log2(gene read count/gene length) and estimated average RNA copies per cell (column N) considering 4 RNA sequencing samples covering different growth times (6, 24 and 48 hours, ArrayExpress identifier E‐MTAB‐6203). Column O accounts for the number of mass spectrometry experiments detecting that entry (to a maximum of 116) and column P accounts for the total number of unique tryptic peptides detected. This comes <a href="https://paperpile.com/c/BImj5N/eljz">[5]</a>, available for 12,426 sequences that present an amino acid length ≥19 (from 116 mass spectrometry experiments, ID PRIDE: PXD008243). Columns Q to T recapitulate protein copies per cell under different conditions (overall, extracting with urea, extracting with SDS and mean, respectively). Column U includes half-lives of the proteins. Columns V and W describe the reference density of insertion and essentiality assigned in previous studies. Column X-AA includes the predicted RanSEPs score, ribosome binding site presence, homology into seven groups: 0—no hits passed the thresholds defined; 1—conserved with an annotated function; 2—conserved as an annotated SEP in NCBI but no associated function; 3—conserved in a different species but target and homologous sequence not found in NCBI; 4—sequence is completely or partially (> 75%) repeated ≥ 3 times in the reference genome; 5—potential pseudogene; and 6—to depict those annotations that are found in the reference NCBI annotation file. , and function expected by homology, respectively. Columns AB to AD cover the output provided by Phobius, including the number of transmembrane segments, presence of signal peptide and transmembrane topology predicted by TM-HMM. Column AE includes the complex information where 1 implies that entry is functional as a monomer, 2 as dimer, and so on. Finally, columns AF-AH will be 1 if the protein is a Lon protease target, a lipoprotein, and/or a truncated gene or pseudogene, respectively, 0 otherwise. Following columns include for every sample presenting selective insertion rates in-frame using the following identifiers separated by underscores: marker (BarnB, Cm or Ery), type (control-AC or selection-BD, antibiotic concentration, sample replicate, frame measured, metric. Metrics account for number insertions in-frame (<em>I</em>), read count (<em>R</em>), linear density from non-coding regions used in the Poisson evaluation (<em>rNC</em>), probability measured (<em>sfNC</em>) and a binary for prediction (<em>pred</em>; 0 - no significant, 1 - significant). Last columns combine the number of samples each annotation has been identified. Notice for barnase library the results need to be interpreted considering it is a negative selection marker inverting the 0 and 1 meaning.</p>
ARPIP: Ancestral sequence Reconstruction with insertions and deletions under the Poisson Indel Process
<p>Modern phylogenetic methods allow inference of ancestral molecular sequences given an alignment and phylogeny relating present day sequences. This provides insight into the evolutionary history of molecules, helping to understand gene function and to study biological processes such as adaptation and convergent evolution across a variety of applications. Here we propose a dynamic programming algorithm for fast joint likelihood-based reconstruction of ancestral sequences under the Poisson Indel Process (PIP). Unlike previous approaches, our method, named ARPIP, enables the reconstruction with insertions and deletions based on an explicit indel model. Consequently, inferred indel events have an explicit biological interpretation. Likelihood computation is achieved in linear time with respect to the number of sequences. Our method consists of two steps, namely finding the most probable indel points and reconstructing ancestral sequences. First, we find the most likely indel points and prune the phylogeny to reflect the insertion and deletion events per site. Second, we infer the ancestral states on the pruned subtree in a manner similar to FastML. We applied ARPIP on simulated datasets and on real data from the Betacoronavirus genus. ARPIP reconstructs both the indel events and substitutions with a high degree of accuracy. Our method fares well when compared to established state-of-the-art methods such as FastML and PAML. Moreover, the method can be extended to explore both optimal and suboptimal reconstructions, include rate heterogeneity through time and more. We believe it will expand the range of novel applications of ancestral sequence reconstruction.</p>
ARPIP: Ancestral sequence Reconstruction with insertions and deletions under the Poisson Indel Process
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Immunization of Patients With Metastatic Melanoma Using the GP100 Peptide Preceded by an Endoplasmic Reticulum Insertion Signal Sequence
ClinicalTrials.gov study NCT00001705. IPD Sharing: Not stated. Countries: 1. Publications: 3.
Data from: High-throughput sequencing of transposable element insertions suggests adaptive evolution of the invasive Asian Tiger Mosquito towards temperate environments
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Data from: Mining for single nucleotide polymorphisms and insertions / deletions in expressed sequence tag libraries of oil palm
The oil palm is a tropical oil bearing tree. Recently EST-derived SNPs and SSRs are a free by-product of the currently expanding EST (Expressed Sequence Tag) data bases. The development of high-throughput methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion / deletion) has led to a revolution in their use as molecular markers. Available (5452) Oil palm EST sequences were mined from dbEST of NCBI. CAP3 program was used to assemble EST sequences into contigs. Candidate SNPs and Indel polymorphisms were detected using the perl script auto_snip version 1.0 which has used 576 ESTs for detecting SNPs and Indel sites. We found 1180 SNP sites and 137 indel polymorphisms with frequency 1.36 SNPs / 100 bp. Among the six tissues from which the EST libraries had been generated, mesocarp had high frequency of 2.91 SNPs and indels per 100 bp whereas the zygotic embryos had lowest frequency of 0.15 per 100 bp. We also used the Shannon index to analyze the proportion of ten possible types of SNP/indels. ESTs from tissues of normal apex showed highest values of Shannon index (0.60) whereas abnormal apex had least value (0.02). The present report deals the use of Shannon index for comparing SNP/ indel frequencies mined from ESTlibraries and also confirm that the frequency of SNP occurrence in oil palm to use them as markers for genetic studies.
Data from: Plastome sequencing of ten nonmodel crop species uncovers a large insertion of mitochondrial DNA in cashew
In plant evolution, intracellular gene transfer (IGT) is a prevalent, ongoing process. While nuclear and mitochondrial genomes are known to integrate foreign DNA via IGT and horizontal gene transfer (HGT), plastid genomes (plastomes) have resisted foreign DNA incorporation and only recently has IGT been uncovered in the plastomes of a few land plants. In this study, we completed plastome sequences for l0 crop species and describe a number of structural features including variation in gene and intron content, inversions, and expansion and contraction of the inverted repeat (IR). We identified a putative rpl22 in cinnamon (Cinnamomum verum J. Presl) and other sequenced Lauraceae and an apparent functional transfer of rpl23 to the nucleus of quinoa (Chenopodium quinoa Willd.). In the orchard tree cashew (Anacardium occidentale L.), we report the insertion of an ∼6.7-kb fragment of mitochondrial DNA into the plastome IR. BLASTn analyses returned high identity hits to mitogenome sequences including an intact ccmB open reading frame. Using three plastome markers for five species of Anacardium, we generated a phylogeny to investigate the distribution and timing of the insertion. Four species share the insertion, suggesting that this event occurred <20 million yr ago in a single clade in the genus. Our study extends the observation of mitochondrial to plastome IGT to include long-lived tree species. While previous studies have suggested possible mechanisms facilitating IGT to the plastome, more examples of this phenomenon, along with more complete mitogenome sequences, will be required before a common, or variable, mechanism can be elucidated.
Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteremia.
<p>Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in dissemination and modification of the antibiotic resistant phenotype. To that end, we leveraged fully resolved <em>Enterococcus faecium</em> and <em>Enterococcus faecalis</em> genomes of isolates collected over five days from an inpatient with prolonged bacteremia. Isolates from both species harbored similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of <em>E. faecium</em> isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in <em>E. faecalis</em> isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in <em>liaSR</em> genes and a putative composite transposon in<em> E. faecium</em>, offering a new paradigm for the study of daptomycin resistance and novel insights into the dissemination of daptomycin resistance. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.</p>
Data from: Mining for single nucleotide polymorphisms and insertions / deletions in expressed sequence tag libraries of oil palm
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Data from: Plastome sequencing of ten nonmodel crop species uncovers a large insertion of mitochondrial DNA in cashew
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High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria
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TrapSeq: An RNA Sequencing-based pipeline for the identification of genetrap insertions in mammalian cells
GEO Series GSE99483. Homo sapiens. 6 samples. Type: Expression profiling by high throughput sequencing.
Sequencing de novo Mu transposon insertions across maize tissues
GEO Series GSE279993. Zea mays. 56 samples. Type: Other.
Characterization of Missing Human Genome Sequences and Copy-number Polymorphic Insertions.
GEO Series GSE20634. Homo sapiens. 29 samples. Type: Genome variation profiling by genome tiling array.
RNA sequencing of seeds from Mthsfa9 Tnt1 insertion mutants
GEO Series GSE154256. Medicago truncatula. 9 samples. Type: Expression profiling by high throughput sequencing.
Alteration of genome folding via engineered transposon insertion [Target-enriched sequencing]
GEO Series GSE137371. Homo sapiens. 2 samples. Type: Other.
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