Dataset Information

EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome.

ABSTRACT: Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12,063 ACEGs (assembly of contiguous ESTs based on genome) and generated 15,857 contigs of average length 934 nt. We predict that roughly 3000 of our contigs represent full-length transcripts. Compared to previous assemblies, ACEGs show extended contig length, increased accuracy and a reduction in redundancy. Because our assembly protocol also uses ESTs with no corresponding genomic sequences, it provides sequence information for genes interrupted by sequence gaps. Detailed analysis of randomly sampled ACEGs reveals several hundred putative cases of alternative splicing, many overlapping transcription units and new genes not identified by gene prediction algorithms. Our protocol, although developed for and tailored to the C. reinhardtii dataset, can be exploited by any eukaryotic genome project for which both a draft genome sequence and ESTs are available.

SUBMITTER: Jain M

PROVIDER: S-EPMC1874618 | biostudies-other | 2007

REPOSITORIES: biostudies-other

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EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome.

Jain Monica M Shrager Jeff J Harris Elizabeth H EH Halbrook Renee R Grossman Arthur R AR Hauser Charles C Vallon Olivier O

Nucleic acids research 20070313 6

Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12,063 AC ...[more]

PMID: 17355987

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Project description:BackgroundGenome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs.ResultsOur analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy.ConclusionsThe extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.

Dataset Information

EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome.

Publications

EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome.

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