| 研究課題/領域番号 |
17J00597
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
BEEDESSEE Girish 沖縄科学技術大学院大学, 科学技術研究科, 特別研究員(DC1)
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| 研究期間 (年度) |
2017-04-26 – 2020-03-31
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| キーワード | dinoflagellates / toxin / genome / transcriptome / polyketide synthase |
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| 研究実績の概要 |
The purpose of this project is to undertake the challenge of sequencing the Amphidinium dinoflagellate genome with higher DNA content and understand how secondary metabolites are produced and for what purposes. The study employs next-generation sequencing as well as several -omics approaches to address these aims.
In order to generate a genome assembly, 12 long insert size libraries ranging from 2 kb to 18 kb were prepared and sequenced using Hiseq 4000. The previous assembled genome was subjected to two rounds of scaffolding using the quality controlled mate-pair reads and this improved the assembly significantly with N50 of 166 kb. This scaffolded Amphidinium genome was checked for genome completeness using BUSCO software, where the presence of 429 highly conserved eukaryotic genes (CEGs) was determined. This resulted in a low completeness BUSCO score of 33.7 %. An alternative approach was to use blast the 458 CEGs from CEGMA software against the Amphidinium genome so as to identify potential homologs at a cutoff value of 1e-5. This resulted in the recovery of 352 (77%) homologs.
The completeness of this transcriptome was assessed using BUSCO and the score was 80.9 %. STAR was used to map RNA-seq reads to the assembled genome. It was found that only 80% reads could be mapped.
In order to understand the localization of enzymes involved in secondary metabolism, confocal microscopy was undertaken using anti-KS antibody (provided by Dr. Van Dolah, NOAA, USA). The enzymes appear to be mostly localized around the cell external surface as well as in chloroplast areas.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
According to the research proposal, nearly 50% of the genome project has been completed and the remaining 50% is expected to be completed by end of this year.
The gene prediction is taking much time because the genome is big and also there are some complicated regions of genes such presence of non-canonical splice site.
Commonly used softwares do not handle such non-canonical sites, so the software needs to be modified at several codes so as to allow such new sites.
So as to solve this problem, I aim to do Isoseq sequencing using Pacbio but such technique is time-consuming and data analysis is difficult.
Transcriptome assembly and annotation have been completed. Such assembly will be helpful is gene prediction.
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| 今後の研究の推進方策 |
1. In order to investigate the effect of nitrate and phosphate depletion stress with respect to secondary metabolism, cultures were setup with different nutrient level and RNA samples were isolated. This study aims to investigate the differential expression of secondary metabolites associated genes. Currently, miRNA libraries and mRNA libraries will be prepared for sequencing. The effect of miRNAs as regulator of mRNA expression will be studied.
2.The genome sequencing has been completed and now gene are being predicted using modified version of software codes.Once genes are predicted, they will be annotated.
3. Mass spectrometry of metabolites will be be undertaken to assess their production using different stress condition.
4. Combining transcriptome data with metabolomics data, pathways for toxin biosynthesis will be surveyed in the genome.
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