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Next Generation Sequencing facilitates analysis of gene expression from Rehmannia glutinosa associated with replanting disease

ABSTRACT: Purpose: the technology of Solexa/Illumina (RNA-seq) is an attractive alternative to the transcriptome sequencing. The goals of this study are to investigate transcriptional changes in replanted R. glutinosa leaves and identify genes responding to the disease. Methods:Totally 6.01and 6.15 million raw tags were measured from leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, respectively. mRNA profiles of leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, were generated by deep sequencing (Illumina HiSeq™ 2000 system). qRT–PCR validation was performed using SYBR Green assays. Results: we obtained a total of 6.01 and 6.15 million total tags including 377,200 and 361,659 distinct tags from the L1 and L2 samples, respectively. Removal of low quality tags, 161,298 and 149,290 distinct clean tags were remained.With reference to the 94,544 non-redundant consensus sequences defined by the RNA-seq transcriptomic procedure, 60,574 genes had the diagnostic CATG site, which were taken as reference distinct sequences for DGE analysis. 47,458 of the L1 tag library and 42,247 of the L2 tag library were perfectly matched the reference sequences. we screened differentially expressed genes in the L1 and L2 libraries by digital gene expression (DGE) technique. Finally, a set of 1,954 genes may be in differential expression of L2. By bioinformatics and qRT-PCR, the 117 most strongly differentially expressed ones were considered to be prime candidates for responsible for replanting disease. Conclusions: The study provides an important resource for further investigating the cause of replanting disease and developing the methods to control or subtract its injury.

ORGANISM(S): Rehmannia glutinosa

PROVIDER: GSE54620 | GEO | 2015/12/31

SECONDARY ACCESSION(S): PRJNA237196

REPOSITORIES: GEO

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Project description:Purpose: the technology of Solexa/Illumina (RNA-seq) is an attractive alternative to the transcriptome sequencing. The goals of this study are to investigate transcriptional changes in replanted R. glutinosa leaves and identify genes responding to the disease. Methods:Totally 6.01and 6.15 million raw tags were measured from leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, respectively. mRNA profiles of leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, were generated by deep sequencing (Illumina HiSeqâ¢ 2000 system). qRTâPCR validation was performed using SYBR Green assays. Results: we obtained a total of 6.01 and 6.15 million total tags including 377,200 and 361,659 distinct tags from the L1 and L2 samples, respectively. Removal of low quality tags, 161,298 and 149,290 distinct clean tags were remained.With reference to the 94,544 non-redundant consensus sequences defined by the RNA-seq transcriptomic procedure, 60,574 genes had the diagnostic CATG site, which were taken as reference distinct sequences for DGE analysis. 47,458 of the L1 tag library and 42,247 of the L2 tag library were perfectly matched the reference sequences. we screened differentially expressed genes in the L1 and L2 libraries by digital gene expression (DGE) technique. Finally, a set of 1,954 genes may be in differential expression of L2. By bioinformatics and qRT-PCR, the 117 most strongly differentially expressed ones were considered to be prime candidates for responsible for replanting disease. Conclusions: The study provides an important resource for further investigating the cause of replanting disease and developing the methods to control or subtract its injury. Leaf mRNA profiles of L1 (The first year planted R. glutinosa leaves ) and L2 (the second year replanted R. glutinosa leaves) root expansion stage were generated by deep sequencing (using Illumina HiSeqâ¢ 2000 system)

Project description:Purpose: Development of resistance to tamoxifen is an important clinical issue in the treatment of patients with breast cancer. Tamoxifen resistance may be the result of the acquisition of epigenetic regulation such as DNA methylation within breast cancer cells resulting in changed mRNA expression of genes being pivotal for estrogen dependent growth. Alternatively, tamoxifen resistance may be due to selection of preexisting resistant cells, which may exhibit cancer stem-like characteristics or a combination of the two mechanisms. Methods: To evaluate the contribution of these possible mechanisms to tamoxifen resistance, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massively parallel sequencing to analyze a well-established tamoxifen resistant cell line model: MCF-7/S0.5 (tamoxifen sensitive parental cell line) and 4 high-dosage tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8). MMSDK uses BssHII as mapping enzyme (DNA methylation sensitive enzyme). Both MMSDK and DGE use NlaIII and MmeI to produce 20-21 bp tag. The indexed single-end sequencing was performed by Illumina HiSeq 2000 in BGI-Shenzhen. A dynamic programming algorithm-FASTX-Toolkit implemented in Perl was used to trim the adaptor sequence. The trimmed tags were subjected to quality filtering, so that only tags with sequencing quality higher than 30 for more than 80% of the nucleotides were used for subsequent analysis. For MMSDK tag mapping, we generated a simulated reference library, i.e., BssHII reference library, by in silico enzyme digestion of the human genome (hg19, UCSC) regardless of the methylation state. This library was used as reference for subsequent mapping of the tags in the MMSDK analysis. In the DGE analysis, refMrna (hg19, UCSC) was used as reference for mapping cDNA tags. Subsequently, the Burrows–Wheeler Aligner (BWA) procedure for aligning the MMSDK and DGE tags to the simulated BssHII reference library and refMrna reference library, respectively, was applied. Results: MMSDK libraries using BssHII/NlaIII were generated from the parental tamoxifen sensitive subline MCF-7/S0.5 and the 4 TAMR cell lines: TAMR-1, TAMR-4, TAMR-7 and TAMR-8. The 5 indexed MMSDK libraries were sequenced in one lane and 1.38 Gb clean tag data for all 5 cell lines were obtained, with an average sequencing amount of ~270 Mb per library. On average, 59.5 % of the tags with mapping quality ≥ 20 were mapped back to the simulated BssHII/NlaIII reference library. DGE libraries were also generated from MCF-7/S0.5 and the 4 TAMR cell lines. The 5 indexed DGE libraries were sequenced in one lane and obtained 1.71 Gb clean tag data for all 5 cell lines with an average sequencing amount of ~340 Mb per library. On average, 40.8 % with mapping quality ≥ 20 were mapped back to the simulated NlaIII human transcriptome (refMrna reference library). Our present study demonstrates large differences in global gene expression and DNA methylation profiles between parental tamoxifen-sensitive cell line and 4 high-dosage tamoxifen treatment selected resistant sublines. The tamoxifen resistant cell lines exhibited globally higher methylation level than the parental cell line and an inverse relationship between gene expression and DNA methylation in the promoter regions were noticed. High expression of SOX2 and alterations of other SOX gene family members, E2F gene family members and RB-related pocket protein genes as well as highlighted stem cell pathways imply that cancer initiating cells/stem cells are involved in the resistance to tamoxifen.

Project description:All above ground organs of higher plants are ultimately derived from specialized organogenic structures termed shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, marked by cell layering. Maize SAMs are comprised of two cell layers, L1 (single cell layered tunica) and L2 (corpus). To identify genes required for layer-specific functions intact maize SAMs were fixed, embedded in paraffin and sectioned. L1 and L2 cells were isolated from these sections via laser capture microdissection (LCM). RNA was isolated from six biological replications of L1 and L2, amplified and hybridized to microarrays spotted with ~37,000 maize cDNA clones. This experiment identified ~700 ESTs that are preferentially expressed in the L1 or the L2 (P <0.001). The L1-up-regulated ESTs included ZmOCL1 and ZmOCL4, which are known to exhibit L1-specific expression in the maize SAM. The L2-up-regulated ESTs included KNOTTED1, whose transcripts are known to accumulate in the L2 but not in the L1 of the maize SAM. Differentially expressed ESTs included genes involved in transcription, signal transduction, transport and metabolism, many of which are novel candidates that are required for layer-specific functions in the maize SAM. Several L1-up-regulated ESTs were annotated as yabby family genes or basic helix-loop-helix transcription factor-like genes, which have not previously been reported as having layer-specific expression in the SAM. Novel WW domain-containing genes (WW genes) were identified in this study. The WW domain mediates protein-protein interactions, often with signal transduction components. These WW genes were substantially up-regulated in the L1 relative to the L2. Keywords: Cell Type Comparison An experimental aim is to identify genes that are differentially expressed in distinct histological cell layers of maize SAM by comparing the transcript accumulation between L1 (single cell layered tunica) and L2 (corpus) using cDNA microarrays that have over 37,000 informative spots from maize.

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Next Generation Sequencing facilitates analysis of gene expression from Rehmannia glutinosa associated with replanting disease

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