|
| Status |
Public on Jun 20, 2017 |
| Title |
Affected coral tissue from Waiopae, Hawaiʻi (Mcap_WA3) |
| Sample type |
SRA |
| |
|
| Source name |
Coral tissue
|
| Organism |
coral metagenome |
| Characteristics |
site: Waiopae, Hawaii
tissue type: Affected
coral colony number: Diseased 3
coral colony health state: Diseased (growth anomaly)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from ~0.1g tissue powder using a combination of TRIzol reagent (Life Technologies) and the RNeasy Mini Kit (Qiagen). TRIzol was added to sample tubes (1ml per 0.1g tissue) and tubes were incubated for 5min at room temperature. Samples were then centrifuged at 12,000g at 4˚C for 10min, and the supernatant transferred to an RNase-free 1.5ml tube. Chloroform (Sigma-Aldrich) was added (0.2ml per 1ml TRIzol), the samples were shaken vigorously for 20–30 seconds, and then incubated at room temperature for 3 minutes. Samples were then centrifuged at 18,000g at 4˚C for 18 minutes. The top layer of aqueous solution was transferred to an RNase-free tube and an equal volume of 200 proof molecular grade ethanol (Sigma-Aldrich) was added. RNA was purified using the RNeasy Mini Kit with a 25min DNA digestion step (Qiagen RNase-free DNase Set) and eluted in 60µl DEPC-treated water (Omega). RNA quality and quantity was determined using the Qubit RNA Broad Range Assay Kit (Life Technologies) and Agilent RNA 6000 Nano Kit (Agilent), and RNA aliquots were stored at –80˚C.
mRNA is purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C. Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries. The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX. Samples with a yield of ≥0.5 ng/ul are used for sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Montipora capitata holobiont
Coral tissue sample from a M. capitata colony affected by the disease growth anomaly. Tissue sampled directly from a growth anomaly lesion.
Metatranscriptome_denovoassembly.fasta
Metatranscriptome_qualityfiltered.fasta
Montiporacapitata_transcriptome.fasta
Metatranscriptome_qualityfiltered_genes_FPKM.tsv
Metatranscriptome_denovoassembly_isoforms_TPM.tsv
Montiporacapitata_transcriptome_genes_FPKM.tsv
Montiporacapitata_transcriptome_isoforms_TPM.tsv
Mcap_WA3
|
| Data processing |
Signal intensities are converted to individual base calls during a run using the system's Real Time Analysis (RTA) software. Sample de-multiplexing is performed using Illumina's CASAVA 1.8.2 software suite.
Trimmomatic (version 0.32) was used to quality filter raw reads. Parameters: WINSIZE=5; WINCUTOFF=20; LEADING=20; TRAILING=20; MINLEN=50
Trinity (version trinityrnaseq_r20140413p1) default parameters were used to construct de novo metatranscriptome assembly using in silico normalized reads (50X coverage; Trinity default settings).
RSEM and edgeR were used to calculate abundance statistics (TPM and FPKM) using default Trinity (trinityrnaseq_r20140413p1) perl scripts for gene expression analysis.
Homolog identification using BlastP (version 2.2.23+) and Inparanoid (version 4.1) along with GC-content analysis was utilized to identify the subset of transcripts that are of coral (Montipora capitata) origin.
Supplementary_files_format_and_content: Metatranscriptome_denovoassembly.fasta is a fasta file containing the de novo assembled metatranscriptome of the Montipora capitata holobiont.
Supplementary_files_format_and_content: Metatranscriptome_qualityfiltered.fasta is a fasta file containing the quality-filtered metatranscriptome of the Montipora capitata holobiont.
Supplementary_files_format_and_content: Montiporacapitata_transcriptome.fasta is a fasta file containing the subset of transcripts from the metatranscriptome that were identified as originating from the coral host (Montipora capitata).
Supplementary_files_format_and_content: Metatranscriptome_qualityfiltered_genes_FPKM.tsv is a tab-delimited text file with TMM normalized FPKM abundance values assessed at the gene (unigene) level for the quality-filtered metatranscriptome. This data was utilized to assess differential gene expression at the metatranscriptome level.
Supplementary_files_format_and_content: Metatranscriptome_denovoassembly_isoforms_TPM.tsv is a tab-delimited text file with raw read count abundance values assessed at the isoform (transcript) level for the entire metatranscriptome. This data was utilized to conduct multidimensional scaling analysis at the metatranscriptome level.
Supplementary_files_format_and_content: Montiporacapitata_transcriptome_genes_FPKM.tsv is a tab-delimited text file with TMM normalized FPKM abundance values assessed at the gene (unigene) level for the coral (Montipora capitata) transcriptome subset. This data was utilized to assess differential gene expression at the coral transcriptome level.
Supplementary_files_format_and_content: Montiporacapitata_transcriptome_isoforms_TPM.tsv is a tab-delimited text file with raw read count abundance values assessed at the isoform (transcript) level for the coral (Montipora capitata) transcriptome subset. This data was utilized to conduct multidimensional scaling analysis at the coral transcriptome level.
Supplementary_files_format_and_content: Metatranscriptome_denovoassembly_isoforms_TPM_kiholo.tsv is a tab-delimited text file with raw read count abundance values assessed at the isoform (transcript) level for the entire metatranscriptome for samples collected at the Kiholo site.
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| |
|
| Submission date |
Apr 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Monika Frazier |
| E-mail(s) |
mfrazier@hawaii.edu
|
| Organization name |
University of Hawaii at Hilo
|
| Department |
Tropical Conservation Biology and Environmental Science
|
| Street address |
200 West Kawili Street
|
| City |
Hilo |
| State/province |
HI |
| ZIP/Postal code |
96720 |
| Country |
USA |
| |
|
| Platform ID |
GPL23310 |
| Series (1) |
| GSE97888 |
De novo metatranscriptome assembly and coral gene expression profile of Montipora capitata with growth anomaly |
|
| Relations |
| BioSample |
SAMN06560382 |
| SRA |
SRX2741916 |
