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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.

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FL-cDNA sequences with 5′ poly(A) tail from plants and animals. The 5′ poly(A) sequences are shown in boldface letters and translation initiation codon (ATG) is shown in capital letters.
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fig4: FL-cDNA sequences with 5′ poly(A) tail from plants and animals. The 5′ poly(A) sequences are shown in boldface letters and translation initiation codon (ATG) is shown in capital letters.

Mentions: Surprisingly, we found poly(A) tails at the 5′ ends of maize transcripts (Figure 3 and Supplementary Figures 1 and 2). Over 8% of the maize transcripts consisted in both G-capping and poly(A) signature in the 5′ regions. The length of the 5′ poly(A) tail varied from 20 to 150 adenine residues in most transcripts (Figure 3 and Supplementary Figures 1 and 2). Except for the rubisco small subunit gene (Supplementary Figure 3), we found poly(A) tails for other highly expressed genes (Figure 3 and Supplementary Figures 1 and 2). To investigate if any FL-cDNAs had 5′ poly(A) tails, we searched plant, animal and viral cDNA databases. Similarly, several FL-cDNAs with 5′ poly(A) tail were found in plants (maize, rice and Arabidopsis), animals (human, mouse and Drosophila) and viruses (vaccine and cowpox virus) (Figure 4). Interestingly, the translation initiation codon (ATG) was followed immediately after the 5′ poly(A) tail in the FL-cDNAs from Arabidopsis, rice, human, mouse, Drosophila and virus, but this feature was not observed in maize FL-cDNAs (Figure 4).


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)

FL-cDNA sequences with 5′ poly(A) tail from plants and animals. The 5′ poly(A) sequences are shown in boldface letters and translation initiation codon (ATG) is shown in capital letters.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: FL-cDNA sequences with 5′ poly(A) tail from plants and animals. The 5′ poly(A) sequences are shown in boldface letters and translation initiation codon (ATG) is shown in capital letters.
Mentions: Surprisingly, we found poly(A) tails at the 5′ ends of maize transcripts (Figure 3 and Supplementary Figures 1 and 2). Over 8% of the maize transcripts consisted in both G-capping and poly(A) signature in the 5′ regions. The length of the 5′ poly(A) tail varied from 20 to 150 adenine residues in most transcripts (Figure 3 and Supplementary Figures 1 and 2). Except for the rubisco small subunit gene (Supplementary Figure 3), we found poly(A) tails for other highly expressed genes (Figure 3 and Supplementary Figures 1 and 2). To investigate if any FL-cDNAs had 5′ poly(A) tails, we searched plant, animal and viral cDNA databases. Similarly, several FL-cDNAs with 5′ poly(A) tail were found in plants (maize, rice and Arabidopsis), animals (human, mouse and Drosophila) and viruses (vaccine and cowpox virus) (Figure 4). Interestingly, the translation initiation codon (ATG) was followed immediately after the 5′ poly(A) tail in the FL-cDNAs from Arabidopsis, rice, human, mouse, Drosophila and virus, but this feature was not observed in maize FL-cDNAs (Figure 4).

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