Limits...
Robust analysis of 5'-transcript ends (5'-RATE): a novel technique for transcriptome analysis and genome annotation.

Gowda M, Li H, Alessi J, Chen F, Pratt R, Wang GL - Nucleic Acids Res. (2006)

Bottom Line: Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes.Sequence analysis of a maize 5'-RATE library revealed complex alternative transcription start sites and a 5' poly(A) tail in maize transcripts.Our results demonstrate that 5'-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 5'-transcript ends (5'-RATE) for rapid and cost-effective isolation of long 5' transcript ends (approximately 80 bp). It consists of three major steps including 5'-oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 5'-RATE library revealed complex alternative transcription start sites and a 5' poly(A) tail in maize transcripts. Our results demonstrate that 5'-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.

Show MeSH

Related in: MedlinePlus

Sequence alignment of the jasmonate-induced gene (ID: Q564C9) with its alternative TSSs and 5′ poly(A) tail. The nucleotides in underlined are non-template sequences.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1636456&req=5

fig3: Sequence alignment of the jasmonate-induced gene (ID: Q564C9) with its alternative TSSs and 5′ poly(A) tail. The nucleotides in underlined are non-template sequences.

Mentions: Preliminary sequence analysis of the 5′-RATE tags revealed that many maize transcripts had alternative TSSs. For example, the gene encoding a jasmonate-induced protein (ID: Q564C9) had 46 different TSSs (Figure 3) and the rubisco small subunit-encoding gene (ID: P05348) had 9 different TSSs (Supplementary Figure 3). In general, the TSS location of different transcripts varied from 1 to 99 nt from the 5′ region of maize FL-cDNAs. The length of the TSSs ranged from 8 to 14 nt (Figure 3 and Supplementary Figures 1–3). The analysis of the TSS data did not reveal any consensus sequences. Among the analyzed transcripts, the rubisco small subunit-encoding gene had the lowest 5′-tag diversity (33.33%) (Supplementary Figure 3). The transcript with the highest tag diversity (90%) was the gene encoding an intermediate filament C2 protein (ID: Q9NG13) with 2–75 non-template derived nucleotides (Supplementary Figure 2). Alternative TSSs were found for a lot of genes analyzed in this study, which is similar with the findings in the FL-cDNA libraries in Arabidopsis (25).


Robust analysis of 5'-transcript ends (5'-RATE): a novel technique for transcriptome analysis and genome annotation.

Gowda M, Li H, Alessi J, Chen F, Pratt R, Wang GL - Nucleic Acids Res. (2006)

Sequence alignment of the jasmonate-induced gene (ID: Q564C9) with its alternative TSSs and 5′ poly(A) tail. The nucleotides in underlined are non-template sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636456&req=5

fig3: Sequence alignment of the jasmonate-induced gene (ID: Q564C9) with its alternative TSSs and 5′ poly(A) tail. The nucleotides in underlined are non-template sequences.
Mentions: Preliminary sequence analysis of the 5′-RATE tags revealed that many maize transcripts had alternative TSSs. For example, the gene encoding a jasmonate-induced protein (ID: Q564C9) had 46 different TSSs (Figure 3) and the rubisco small subunit-encoding gene (ID: P05348) had 9 different TSSs (Supplementary Figure 3). In general, the TSS location of different transcripts varied from 1 to 99 nt from the 5′ region of maize FL-cDNAs. The length of the TSSs ranged from 8 to 14 nt (Figure 3 and Supplementary Figures 1–3). The analysis of the TSS data did not reveal any consensus sequences. Among the analyzed transcripts, the rubisco small subunit-encoding gene had the lowest 5′-tag diversity (33.33%) (Supplementary Figure 3). The transcript with the highest tag diversity (90%) was the gene encoding an intermediate filament C2 protein (ID: Q9NG13) with 2–75 non-template derived nucleotides (Supplementary Figure 2). Alternative TSSs were found for a lot of genes analyzed in this study, which is similar with the findings in the FL-cDNA libraries in Arabidopsis (25).

Bottom Line: Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes.Sequence analysis of a maize 5'-RATE library revealed complex alternative transcription start sites and a 5' poly(A) tail in maize transcripts.Our results demonstrate that 5'-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 5'-transcript ends (5'-RATE) for rapid and cost-effective isolation of long 5' transcript ends (approximately 80 bp). It consists of three major steps including 5'-oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 5'-RATE library revealed complex alternative transcription start sites and a 5' poly(A) tail in maize transcripts. Our results demonstrate that 5'-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.

Show MeSH
Related in: MedlinePlus