Targeted NGS of circulating tumor DNA
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB12671 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR1254340.fastq.gz | Fastqsanger.gz | |||
| ERR1254350.fastq.gz | Fastqsanger.gz | |||
| ERR1254360.fastq.gz | Fastqsanger.gz | |||
| ERR1254370.fastq.gz | Fastqsanger.gz | |||
| ERR1254380.fastq.gz | Fastqsanger.gz |
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2013-09-08 | E-MTAB-1794 | 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
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.
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Project description:Purpose: The goals of this study are to determine the effect of microRNA-17 overexpression on 20,803 human genes in RASFs using Ion ProtonTM System platform. Human RASFs from two RA patients were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared using miRNeasy kit (Qiagen). Total RNA integrity was checked using an Agilent Technologies 2100 Bio analyzer (Santa Clara, CA). 10 ng of high quality RNA was used to make cDNA for amplification with the Ion AmpliSeq Transcriptome Human Gene Expression kit (ThermoFisher Scientific). The cDNA was subjected to 12 cycles of amplification with panel primers and barcoded with adapters as recommended. Resulting sequencing libraries were quantified by qPCR using SYBR FAST master mix from KapaBiosystems (Wilmington, MA). Sets of eight libraries were balanced, pooled and sequencing beads produced on an Ion Chef. Sequencing was performed on an Ion P1 semi-conductor sequencing chip using an Ion Proton⢠System (ThermoFisher Scientific, Grand Island, NY). Data was collected and primary analysis performed using Torrent Suite software version 5.0.3. Reads were mapped to the panel and expression values determined. R Software version R-3.2.3 was used to generate heatmap. Among the panel of 20,803 genes, the expression of 15,067 genes as shown in the representative heat map was observed in pre-miR-17 and NC-pre-miR transfected RASFs. A total of 664 significantly modulated genes (301 upregulated and 363 downregulated) using Student âtâ test were further utilized for the IPA analysis. The result of IPA predicted the protein ubiquitin pathway as a major canonical pathway affected by the differentially regulated genes. Interestingly, IPA analysis generated an interactome that showed connectivity among various ubiquitin ligases, NF-ÔB family, AP-1/cJun, 20S and 26S proteasome system. Conclusion: Our results clearly shows the major pathways affected by miR-17 overexpression in RASFs were Protein ubiquitination related. mRNA profiles of pre-miR-17 and NC-pre-miR transfected RASFs were generated by AmpliSeq, in duplicate, using Ion Proton⢠System.
2016-09-07 | E-GEOD-83930 | biostudies-arrayexpress
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Project description:Binge drinking contributes to a rising number of emergency room visits in the United States. Our previous work demonstrated that binge drinking impairs cardiac performance and exerts complex hemodynamic alterations through activation of the endocannabinoid–CB1 receptor (CB1R) signaling pathway. Anandamide, an endogenous CB1R agonist, is produced in response to stimuli such as endotoxin challenge and oxidative stress; however, the effect of binge drinking on myocardial anandamide production and subsequent CB1R-mediated cardiodepression remains unclear. Here, we studied the impact of endotoxins derived from intestinal Gram-negative bacteria on myocardial anandamide formation and CB1R-dependent cardiac dysfunction following acute alcohol intoxication. Using a mouse model of binge drinking (5 g/kg orally), we observed a reduced rate of mesenteric microcirculation concurrent with elevated circulating endotoxin levels. Selective intestinal decontamination significantly lowered circulating endotoxin levels, coinciding with reduced cardiac anandamide levels at 3 hours post-binge. These changes were paralleled with improved left ventricular performance, vascular tone, and reduced myocardial oxidative stress. Moreover, cardiac RNA sequencing revealed marked alterations in gene expression following alcohol gavage that were absent in mice with prior selective intestinal decontamination. These findings provide novel mechanistic insights that highlight the crucial role of intestinal Gram-negative bacteria in modulating cardiac anandamide production after binge drinking, thereby exacerbating cardiovascular function and blood redistribution. Cardiac tissues were homogenized in Trizol (Invitrogen, Carlsbad, CA, USA), and total RNA was isolated with a Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop 2000 Spectrophotometer (Thermo Scientific). AmpliSeq libraries were prepared using the Ion AmpliSeq™ Library Kit Plus (Thermo Fisher Scientific, 4488990) per the manufacturer’s instructions. Library quality was determined using the Agilent High Sensitivity DNA Kit (Agilent Technologies, 5067-4626), and library concentrations were quantified using the Ion Library TaqMan™ Quantitation Kit (Life Technologies, 4468802), per manufacturer’s instructions. Barcoded AmpliSeq libraries were loaded on the sequencing chips using the Ion 540TM Chip Kit (ThermoFisher, A27766) and Ion Chef™ Instrument. Libraries were sequenced on the Ion Torrent S5 Sequencing System via the Ion Torrent 540-OT2 kit (ThermoFisher, A27753). 8 to 9 libraries were loaded per sequencing chip, and 2 sequencing ships were loaded and sequenced at a time. An average of 8.5 million counts per sample were sequenced. Alignment and gene expression count were performed using Ion Torrent AmpliSeq RNA Plugin v0.5.4.0 (Thermo Fisher) using mm10 genome.
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