Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1126252 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29478154_subreads.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of plants. The goals of this study are to compare omparatively evaluated both sequence variation and gene expression at the transcriptomic level between two species. Methods: Pooled total RNA of P. floridana flower buds and young fruits, in triplicate, using Illumina HiSeqTM 2000. The sequence reads remove reads with adaptors or unknown nucleotides larger than 5% and low-quality reads using . qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Sequencing the Physalis transcriptome revealed 147,118 unigenes. When aligned to the tomato genome, we estimated that around 30,121 genes were expressed in the Physalis floral-fruit transcriptome, and 10,498 orthologous gene pairs were identified between P. floridana and S. pimpinellifolium.with a fold change ≥1.5 and FER value <0.001, 0.68% of the unigenes in the Physalis floral-fruit transcriptome were developmentally regulated at the floral-fruit transition, and Altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of floral-fruit transcriptomes, with biologic replicates, generated by RNA-seq technology.The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a organ or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2018-12-29 | GSE92998 | GEO
Project description:Aim: We aim to compare current (MeDIP-seq), new (Illumina Infinium 450K BeadChip) and future (PacBio) methods for whole genome DNA methylation analysis. As the interest in determination of disease methylation profiles increases, the scope, advantages and limitations of these methods requires assessment. There are key questions to answer and specific challenges to overcome. For example, how much detail/resolution is sufficient to identify regions of differential methylation and regions of biological/medical significance within a sample? How much coverage of the genome is required for accurate methylation analysis? Is it important to confirm which regions of the genome are unmethylated in addition to focusing on those that are methylated? Loss of methylation may be of equal importance within the cell since this may also contribute to disease pathogenesis. A multi-method (affinity enrichment/bisulphite-conversion based/direct sequencing of methyl-cytosine) and technology platform (Illumina HiSeq/PacBio/Illumina Infinium BeadChip) comparison will enable us to determine the strengths and weakness of each method. We propose to compare four methods using two DNA samples from the Coriell Institute for Cell Repository to assess both current and future capabilities for whole genome methylation analysis in parallel: A) MeDIP-seq using Illumina HiSeq B) Illumina Infinium HumanMethylation 450K BeadChip and C) whole genome methylation sequencing using PacBio. Existing single molecule deep bisulphite sequencing data generated previously from these same samples at the WTSI for targeted regions (30-40 genes) on the human X chromosome will be used to assess performance of each method. The methods selected for this study will generate data covering a range of resolutions from a whole genome scan to array (target defined) resolution and up to single base pair, single molecule resolution; the highest level of detail possible with methods currently available.Samples: DNA from sibling pair GM01240 (female) and GM01240 (male).Requirements: Both samples will be analysed using;A.MeDIP-seq using Illumina HiSeq (one HiSeq lane, 75bp paired end, per sample) B.Illumina Infinium HumanMethylation 450K BeadChipWe are expecting a potentially unnecessary high coverage using one HiSeq lane per sample. However, for the MeDIP procedure we do not have a multiplexing procedure in place. Our requirements for PacBio sequencing have been discussed with and will be supported by the Sequencing Technology Development group.
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