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| Status |
Public on Apr 24, 2020 |
| Title |
Next-Generation Sequencing Revealed L-Carnitine Rescues Congenital Myopathy in Transgenic Zebrafish with Tropomyosin 3 Novel Mutation |
| Organism |
Danio rerio |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: Congenital myopathies (CM) are a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A novel and de novo mutation in tropomyosin 3 [TPM3(E151G)] was identified from a 7-year-old boy with clinically diagnosed CM. TPM3(E151A) mutation was previously reported as causing CM. In this study, we created transgenic zebrafish to investigate the pathogenicity and find potential treatment. The goals of this study are to describe the RNA contents in muscle specimens derived from TPM3(WT), TPM3(E151A), TPM3(E152G) at embryos and adult stages and to compare the differences TPM3(E151A), TPM3(E151G) mutants and TPM3(WT). The biological function of L-carnitine treatment TPM3(E151G) versus without treatment are also predicted and evaluated.
Methods: Muscle mRNA profiles of 6 months adult zebrafish from TPM3(WT), TPM3(E151A) and TPM3(E151G) were extracted and performed deep sequencing, in duplicate, using Illumina Novaseq6000. We also obtained the muscle mRNA profiles of 2 day's zebrafish embryos from TPM3(WT), TPM3(E151A), TPM3(E151G) and TPM3(E151G) treated with L-carnitine. RNA profiles of adult and embryos were generated by deep sequencing, using Illumina NovaSeq 6000.
Results: After acquiring of clean reads, we mapped about 40 million sequence reads per sample to the zebrafish genome (GRCz11). Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with zebrafish genome on a chromosomal level. The quantification of transcript intensity was analyzed using Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM).
Conclusions: Our study represents the first detailed analysis of zebrafish muscle 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 between TPM3(WT), TPM3(E151A) and TPM3(E151G) and L-carnitine treatment. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
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| Overall design |
Next-generation sequencing experiment was performed by NHRI Genomics Core Facility. Sequencing libraries 1 µg total RNA were generated using RNA HyperPrep kit with RiboErase (KAPA Biosystems, Pleasanton, CA, USA), and sequenced on NovaSeq 6000 instrument (Illumina, San Diego, CA, USA). All RNA sample preparation procedures were carried out according to the Illumina's official protocol.
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| Citation(s) |
33435938 |
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| Submission date |
Apr 23, 2020 |
| Last update date |
Jan 19, 2021 |
| Contact name |
Chiou-Hwa Yuh |
| E-mail(s) |
chyuh@nhri.edu.tw
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| Phone |
886-0912281578
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| Organization name |
National Health Research Institutes
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| Street address |
#35 Keyan Road
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| City |
Zhunan Town |
| State/province |
Miaoli County |
| ZIP/Postal code |
35053 |
| Country |
Taiwan |
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| Platforms (1) |
| GPL24995 |
Illumina NovaSeq 6000 (Danio rerio) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA627773 |
| SRA |
SRP258200 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE149261_All_data.xlsx |
8.0 Mb |
(ftp)(http) |
XLSX |
| GSE149261_adult_E151A_vs_WT_FC2X.xlsx |
4.0 Mb |
(ftp)(http) |
XLSX |
| GSE149261_adult_E151G_vs_E151A_FC2X.xlsx |
3.9 Mb |
(ftp)(http) |
XLSX |
| GSE149261_adult_E151G_vs_WT_FC2X.xlsx |
4.1 Mb |
(ftp)(http) |
XLSX |
| GSE149261_embryo_E151A_vs_WT_FC2X.xlsx |
3.8 Mb |
(ftp)(http) |
XLSX |
| GSE149261_embryo_E151G_L_vs_E151G_FC2X.xlsx |
3.8 Mb |
(ftp)(http) |
XLSX |
| GSE149261_embryo_E151G_L_vs_WT_FC2X.xlsx |
3.8 Mb |
(ftp)(http) |
XLSX |
| GSE149261_embryo_E151G_vs_WT_FC2X.xlsx |
3.8 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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