Dataset Information

Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology.

ABSTRACT: Sequencing of the chloroplast (cp) genome using traditional sequencing methods has been difficult because of its size (>120?kb) and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the cp genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassicarapa accessions with one lane per accession. In total, 246, 362, and 361?Mb sequence data were generated for the three accessions Chiifu-401-42, Z16, and FT, respectively. Micro-reads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7-99.8 or 95.5-99.7% of the B. rapa cp genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of cp genome.

SUBMITTER: Wu J

PROVIDER: S-EPMC3492724 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology.

Wu Jian J Liu Bo B Cheng Feng F Ramchiary Nirala N Choi Su Ryun SR Lim Yong Pyo YP Wang Xiao-Wu XW

Frontiers in plant science 20121108

Sequencing of the chloroplast (cp) genome using traditional sequencing methods has been difficult because of its size (>120 kb) and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the cp genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassicarapa accessions with one lane per accession. In total, 246, 362, and 361 Mb sequence data were generated for the t ...[more]

PMID: 23162558

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Project description:BACKGROUND: Downy mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease. RESULTS: Approximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism. CONCLUSIONS: This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.

Project description:Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States. MicroRNAs (miRNAs) play important roles in the regulation of almost every biological process in eukaryotes; however, there is little information available concerning miRNAs in channel catfish. In this study, a small-RNA cDNA library was constructed from 10 tissues of channel catfish, and Solexa sequencing technology was used to perform high-throughput sequencing of the library. A total of 14,919,026 raw reads, representing 161,288 unique sequences, were obtained from the small-cDNA library. After comparing the small RNA sequences with the RFam database, 4,542,396 reads that represent 25,538 unique sequences were mapped to the genome sequence of zebrafish to perform distribution analysis and to screen for candidate miRNA genes. Subsequent bioinformatic analysis identified 237 conserved miRNAs and 45 novel miRNAs in the channel catfish. Stem-loop RT-PCR was applied to validate and profile the expression of the novel miRNAs in 10 tissues. Some novel miRNAs, such as ipu-miR-129b, ipu-miR-7562 and ipu-miR-7553, were expressed in all tissues examined. However, some novel miRNAs appear to be tissue specific. Ipu-miR-7575 is predominantly expressed in stomach. Ipu-miR-7147 and ipu-miR-203c are highly expressed in heart, but are relatively weakly expressed in other tissues. Based on sequence complementarity between miRNAs and mRNA targets, potential target sequences for the 45 novel miRNAs were identified by searching for antisense hits in the reference RNA sequences of the channel catfish. These potential target sequences are involved in immune regulation, transcriptional regulation, metabolism and many other biological functions. The discovery of miRNAs in the channel catfish genome by this study contributes to a better understanding of the role miRNAs play in regulating diverse biological processes in fish and vertebrates.

Dataset Information

Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology.

Publications

Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology.

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