Ena-DATASET-VPC-07-12-2018-20:52:13:493-337 - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001004503 | EGA |
REPOSITORIES: EGA
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Project description:CD3+ T cells were enriched using the EasySep human T cell isolation kit (Stem cell technology). T cells from normal controls or patients with CARD9 mutations were co-cultured with monocytes of one normal control in the presence of heat killed candida. After 3 days, T cell were enriched again to remove the monocytes and total RNA was extracted from the T cells for the RNA-sequencing (RNASeq) evaluation. Targeted RNA sequencing library preparation was carried out using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies), which profiles more than 20,000 human genes; each amplicon (~150 bp) represents a unique targeted gene (one transcript per gene). For library preparation, each sample was run in duplicate, and a cDNA library was generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified with Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified with Agilent Bioanalyzer high-sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with 4 individual samples per pool. Pooled libraries were amplified and enriched with the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies) with Ion PI HiQ kit and chip version 3. We performed gene-level differential expression analysis of targeted RNASeq data using R (v.3.5.3) and the Bioconductor packages DESeq2 (v.1.22.2).
2021-08-12 | GSE181718 | GEO
Project description:Background: Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeqTM Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq.To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Results: Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson’s r=0.92) and Ion Torrent Proton (Pearson’s r=0.92). We used ROC, Matthew’s correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. Conclusions: Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.
2016-02-11 | GSE74760 | GEO
Project description:Purpose: Cystic fibrosis (CF)-associated liver disease (CFLD) is a hepatobiliary complication of CF. Current diagnostic modalities rely on non-specific assessments, while liver biopsy is the gold standard to assess severity of disease. Using miRNA-sequencing we investigated the use of serum miRNA to diagnose and monitor CFLD progression in children with CF. Methods: This was a cross-sectional cohort study of the circulatory miRNA signature of 90 children grouped by clinical, biochemical and imaging assessments as follows: healthy controls (n=30), CF patients with no evidence of liver disease (CFnoLD, n=30) and CFLD (n=30). Serum miRNAs were analysed by miRNA-sequencing. Following total circulatory RNA extraction, RNA samples were pooled together to create three libraries (i.e., 10 patients/library) per study cohort. Libraries were prepared using the Ion Total RNA-Seq Kit v2 protocol (Thermo Fisher, Revision E) and sequenced on the Ion Proton System, using the Ion PI Chip v3 following the Ion PI Hi-Q Sequencing 200 Kit protocol (Thermo Fisher, Revision C). Selected differentially expressed serum miRNA candidates were further validated in 124 individual children serum samples by qRT-PCR and statistical analysis performed to evaluate utility to predict CFLD and hepatic fibrosis severity validated by liver biopsy. Results: The nine libraries sequenced by the Ion Proton System produced 64,658,545 total reads. During processing of the sequencing data, we noticed significant low counts mapped from the CFnoLD library 3. Following principal component analysis this library was confirmed to be an outlier and excluded from the analysis. For the remaining libraries, initial threshold filtering was performed by discarding miRNAs with 10 or less counts and normalized for compositional bias in sequenced libraries and library size. After threshold filtering, 659 miRNAs were identified with a minimum average count of 10.
2018-07-27 | GSE111754 | GEO
Project description:we aimed to compare the mRNA profile between HUVECs treat with HeLa exosome and Hcer cell exosome. Cells were treated with HeLa exosomes or Hcer exosomes for 48 hours (3 repeat samples for each group) and then RNA of each group was collected.RNAs were sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies).According to the results,we found a total of 2493 different genes reach to 2 fold difference between two groups.
2020-02-26 | GSE128802 | GEO
Project description:Evaluation of AmpliSeq and SureSelect whole exome sequencing methods on Ion Proton
| PRJNA481671 | ENA
Project description:We aimed to compare the exosomal microRNA profile form HeLa cell line with that from human cervical epithelial cell(Hcer)line.exosomes were isolated from conditioned medium of HeLa cell and HCer cell,3 samples for each group.Exosomal microRNAs were sequenced on Proton Sequencers according to Ion PI Hi-Q Sequencing 200 Kit (Life Technologies).According to the results of miRNA sequencing, we found a total 371 different miRNAs the amount of which reach to 10 fold differece between two groups.
2020-02-26 | GSE128803 | GEO
Project description:Evaluation of the performance of AmpliSeq and SureSelect exome sequencing libraries for Ion Proton
| PRJEB27429 | ENA
Project description:Five libraries from 100 HEK293 cells each were prepared using a Smartseq based custom library preparation approach with unique molecular identifiers. One batch of 2 replicates (A) and one batch of 3 replicates (B) were prepared from different cell cultures. Libraries were sequenced on an Ion Proton
2016-12-19 | GSE79123 | GEO
Project description:We performed shallow whole genome sequencing (WGS) on circulating free (cf)DNA extracted from plasma or cerebrospinal fluid (CSF), and shallow WGS on the tissue DNA extracted from the biopsy in order to evaluate the correlation between the two biomaterials. After library construction and sequencing (Hiseq3000 or Ion Proton), copy number variations were called with WisecondorX.
2021-11-17 | E-MTAB-10548 | biostudies-arrayexpress
Project description:Transcriptomic profile of human adipose tissue progenitor cells was performed as follows. For AmpliSeq transcriptome sequencing library construction, AmpliSeq™ Library PLUS, AmpliSeq Transcriptome Human Gene Expression Panel and AmpliSeq CD indexes SetA kits were purchased from Illumina and sequencing libraries were constructed as described in AmpliSeq for Illumina Transcriptome Human Gene Expression Panel reference guide (Illumina). Equimolar concentrations of libraries were pooled at 4 nM and denatured and diluted as described in Denature and Dilute Libraries Guide (Illumina) and adjusted to final concentration of 1.4 pM. Resulting library was sequenced on NextSeq 500 using NextSeq 500/550 High Output v2 kit with 2 X 151 bp cycle. Generated raw files were converted to FASTQ files and used for data analysis. AmpliSeq transcriptome FASTQ files were analyzed on Array studio V10.0 (Omicsoft, Qiagen). Following raw read QC, first and last 10 bases were trimmed and mapped to reference genome Human.B38. The read count data was generated using GeneModel RefGene20170606. Resulting data was normalized by DESeq package, transformed to log2 value and used for ANOVA analyses.
2023-01-12 | GSE222749 | GEO