Dataset Information

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology.

ABSTRACT: Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified approximately 120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligated to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate references. Assemblies for these eight genomes are estimated at 88-94% complete, with an average sequence depth of 55x to 186x. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity.

SUBMITTER: Cronn R

PROVIDER: S-EPMC2577356 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology.

Cronn Richard R Liston Aaron A Parks Matthew M Gernandt David S DS Shen Rongkun R Mockler Todd T

Nucleic acids research 20080827 19

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified approximately 120 kb plastomes from e ...[more]

PMID: 18753151

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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.

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.

Dataset Information

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology.

Publications

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology.

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