Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiple insert size paired-end sequencing for deconvolution of complex transcriptomes

ABSTRACT: Deep sequencing of transcriptomes allows quantitative and qualitative analysis of many RNA species in a sample, with parallel comparison of expression levels, splicing variants, natural antisense transcripts, RNA editing and transcriptional start and stop sites the ideal goal. By computational modeling, we show how libraries of multiple insert sizes combined with strand-specific, paired-end (SS-PE) sequencing can increase the information gained on alternative splicing, especially in higher eukaryotes. Despite the benefits of gaining SS-PE data with paired ends of varying distance, the standard Illumina protocol allows only non-strand-specific, paired-end sequencing with a single insert size. Here, we modify the Illumina RNA ligation protocol to allow SS-PE sequencing by using a custom pre-adenylated 3’ adaptor. We generate parallel libraries with differing insert sizes to aid deconvolution of alternative splicing events and to characterize the extent and distribution of natural antisense transcription in C. elegans. Despite stringent requirements for detection of alternative splicing, our data increases the number of intron retention and exon skipping events annotated in the Wormbase genome annotations by 127 % and 121 %, respectively. We show that parallel libraries with a range of insert sizes increase transcriptomic information gained by sequencing and that by current established benchmarks our protocol gives competitive results with respect to library quality.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE40507 | GEO | 2012/08/31

SECONDARY ACCESSION(S): PRJNA174192

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Multiple insert size paired-end sequencing for deconvolution of complex transcriptomes

Project description:Deep sequencing of transcriptomes allows quantitative and qualitative analysis of many RNA species in a sample, with parallel comparison of expression levels, splicing variants, natural antisense transcripts, RNA editing and transcriptional start and stop sites the ideal goal. By computational modeling, we show how libraries of multiple insert sizes combined with strand-specific, paired-end (SS-PE) sequencing can increase the information gained on alternative splicing, especially in higher eukaryotes. Despite the benefits of gaining SS-PE data with paired ends of varying distance, the standard Illumina protocol allows only non-strand-specific, paired-end sequencing with a single insert size. Here, we modify the Illumina RNA ligation protocol to allow SS-PE sequencing by using a custom pre-adenylated 3M-bM-^@M-^Y adaptor. We generate parallel libraries with differing insert sizes to aid deconvolution of alternative splicing events and to characterize the extent and distribution of natural antisense transcription in C. elegans. Despite stringent requirements for detection of alternative splicing, our data increases the number of intron retention and exon skipping events annotated in the Wormbase genome annotations by 127 % and 121 %, respectively. We show that parallel libraries with a range of insert sizes increase transcriptomic information gained by sequencing and that by current established benchmarks our protocol gives competitive results with respect to library quality. Sequencing of mRNA from C. elegans with libraries of four differing insert sizes

2012-08-31 | E-GEOD-40507 | biostudies-arrayexpress

Multiple bacterial species

Project description:Illumina paired-end sequencing with varying library average insert sizes

| PRJNA293409 | ENA

Comparative genomic hybridization of plasmids containing genomic library clones grown in E. coli to monitor copy number changes over time

Project description:We report a genome-wide, multi-scale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves i) growth selections on a mixture of several different plasmid based genomic libraries of defined insert sizes or SCALEs, ii) micro-array studies of enriched plasmid DNA, and a ii) mathematical multi-scale analysis (MSA) that precisely identifies the relevant genetic elements. This approach allows for identification of all single open reading frames and larger multi-gene fragments within a genomic library that alter the expression of a given phenotype. We have demonstrated this method in E. coli by monitoring, in parallel, a population of >106 genomic library clones of different insert sizes, throughout continuous selections over a period of 100 generations.

2006-10-26 | E-TABM-143 | biostudies-arrayexpress

Analysis and design of RNA sequencing experiments for identifying mRNA isoform regulation

Project description:Through alternative splicing, most human genes express multiple isoforms that may have distinct or even antagonistic functions. To infer isoform regulation based on data from high-throughput sequencing of cDNA fragments (RNA-Seq), we have developed MISO, a computational model that estimates the expression level of alternatively spliced exons and mRNA isoforms and provides intuitive measures of con?dence in these estimates. Incorporation of the length distribution of inserted cDNA fragments in paired-end RNA-Seq analysis in MISO enables dramatic improvements in estimation of alternative splicing levels relative to previous methods. We show that one lane of paired-end RNA-Seq data can provide far more information about splicing than two lanes of single-end data, depending critically on properties of the distribution of cDNA fragment lengths in the sequenced library. MISO also leads to an intuitive method to detect di?erentially regulated exons or isoforms. Application of this method implicates the RNA splicing factor hnRNP H in regulation of alternative cleavage and polyadenylation, a role that is supported by UV crosslinking/immunoprecipitation/high-throughput sequencing (CLIP-Seq) analysis. Together, our results provide a probabilistic framework for RNA-Seq analysis, derive functional insights into pre-mRNA processing, and yield guidelines for the optimal design of RNA-Seq experiments for studies of gene and isoform expression. CLIPseq of hnRNP H in HEK 293T cells. RNAseq of polyA+ RNA from C2C12 mouse myoblasts stably expressing an empty vector or a vector containing an shRNA against CUGBP1. Libraries of two different insert lengths were created and examined.

2010-11-07 | E-GEOD-23694 | biostudies-arrayexpress

Transcriptome analysis of PE-degrading Pseudomonas aeruginosa

Project description:Four purified RNA extracts (2 from cells grown in glucose supplemented medium and another 2 from cells grown in PE supplemented medium) were subjected to sequencing. Following cDNA library preparations, the final sequencing libraries were quantified using the KAPA kit (KAPA Biosystem, USA) on a Stratagene Mx-3005P qPCR system (Agilent Technologies, USA) and the respective library sizes were confirmed using Agilent Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies, USA). The resulting libraries were subjected to cluster generation and sequenced using an Illumina flow cell, 202 cycles (101 bp paired-end reads) on the Illumina HiSeq 2000 system (Illumina, USA).

2017-12-19 | E-MTAB-4182 | biostudies-arrayexpress

The impact of Prp18p on splicing fidelity and efficiency in budding yeast

Project description:Fidelity of 3´-splice site (3´SS) selection by the spliceosome is critical for proper gene expression but is a daunting task considering the low complexity of the 3´SS consensus YAG. Here we show that loss of the splicing factor Prp18p in budding yeast activates a diverse array of alternative 3´SS at more than half of all introns. Some alternative sites highly diverge from the YAG consensus, demonstrating a critical role for Prp18p in promoting spliceosome fidelity. Usage of alternative 3´SS is determined by distance from the branchpoint, local RNA secondary structures, upstream poly(U) content, and adenosine enrichment in exons. The 3´SS fidelity function of Prp18p can be genetically uncoupled from its role in splicing efficiency and is promoted by interactions with Slu7p and Prp8p. Taken together, these results provide a comprehensive mechanism into how the spliceosome achieves specificity of 3´SS selection and how it prevents aberrant activation of non-canonical splice sites.

2020-05-25 | GSE131797 | GEO

Sheep Tissue Atlas

Project description:Sheep total RNA was extracted from embryonic and adult tissues. Sequencing libraries were prepared from the RNA using the Illumina TruSeq stranded total RNA with the Ribo Zero gold option for the rRNA removal. The fragmentation in the standard protocol was modified to increase the average insert size in the library. Sequencing with 151 base paired end reads was performed on an Illumina HiSeq 2500 in rapid mode.

2014-12-04 | E-MTAB-3838 | biostudies-arrayexpress

Translational profiling of RBMX-overexpressed HEK293 cells

Project description:Wild-type and RBMX-overexpressed HEK293 cells where profiled at the transcriptome and translatome levels through polysomal profiling. Paired-end RNA-seq libraries were built and sequenced on an Illumina HiSeq 2000 machine to allow for accurate gene expression quantification and alternative splicing isoforms detection.

2019-12-01 | GSE68990 | GEO

Affymetrix exon array analysis of control and hnRNP H knockdown in 293T cells

Project description:Splice site strength and the presence of splicing regulatory elements (SREs) play central roles in the control of pre-mRNA splicing1-3 and in exon evolution4. However, the degree to which SRE function is dependent or independent of the strength of nearby splice sites is not well understood. Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, with important functional and evolutionary implications. Using extensive splicing reporter assays, we found that activity of identical 'G-run' intronic splicing enhancers (ISEs)5, 6 was higher by ~4-fold for 5'ss of intermediate strength relative to weak 5'ss, and higher by ~1.3-fold relative to strong 5'ss, based on established 5'ss scoring criteria7. This dependence on 5'ss strength was supported both by comparative genomic analyses and by high-throughput transcriptome sequencing analyses of splicing changes following RNAi against the G-run-binding factor heterogeneous nuclear ribonucleoprotein (hnRNP) H. This pattern and an inverse pattern of 5'ss-dependent activity observed for G-run exonic splicing silencers (ESSs) enables G-runs and hnRNP H to buffer 5'ss mutations and to function as effective evolutionary capacitors8 of splicing change. Human exons flanked by G-runs exhibited increased 5'ss polymorphism and a ~30% increased frequency of evolutionary change between constitutive and alternative splicing, supporting a role in facilitating splicing-level evolution. Evolutionary conservation indicated a substantially greater role for intronic elements generally in splicing of exons with weak and intermediate 5'ss, and supported 5'ss strength-dependent activity for several other intronic motifs, suggesting widespread functional and evolutionary differences between exons of differing 5'ss strength. Keywords: gene expression array-based, exon and protein coding gene analysis

2009-09-13 | GSE12386 | GEO

Novel SF3B1 Deletion Mutations Result in Aberrant RNA Splicing in CLL Patients

Project description:Recurrent mutations in RNA splicing factors SF3B1, U2AF1, and SRSF2 have been reported in hematologic cancers including myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). However, SF3B1 is the only splicing associated gene to be found mutated in CLL and has been shown to induce aberrant splicing. To investigate if any other genomic aberration caused similar transcriptome changes, we clustered RNASeq samples based on an alternative 3’ splice site (ss) pattern previously identified in SF3B1-mutant CLL patients. Out of 215 samples, we identified 37 (17%) with alternative 3’ ss usage, the majority of which harbored known SF3B1 hotspot mutations. Interestingly, 3 patient samples carried previously unreported in-frame deletions in SF3B1 around K700, the most frequent mutation hotspot. To study the functional effects of these deletions, we used various minigenes demonstrating that recognition of canonical 3’ ss and alternative branchsite are required for aberrant splicing, as observed for SF3B1 p.K700E. The common mechanism of action of these deletions and substitutions result in similar sensitivity of primary cells towards splicing inhibitor E7107. Altogether, these data demonstrate that novel SF3B1 in-frame deletion events identified in CLL result in aberrant splicing, a common biomarker in spliceosome-mutant cancers.

2017-06-27 | GSE95352 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data