Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing

Shibata, Yûkô; Carninci, Piero; Sato, Kenjiro; Hayatsu, Norihito; Shiraki, Toshiyuki; Ishii, Yoshiyuki; Arakawa, Takahiro; Hara, Ayako; Ohsato, N.; Izawa, Masaki; Itoh, Masayoshi; Shibata, Kazuhiro; Shinagawa, Akira; Kawai, Jun; Ota, Yoshimi; Kikuchi, Shoshi; Muramatsu, Masami; Hayashizaki, Yoshihide

doi:10.2144/01315st04

Cited by 12 publications

(10 citation statements)

References 14 publications

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“…The homopolymeric tracks of A/T in the plasmid might inhibit the replication and gene expression processes as shown in Escherichia coli ( 44 , 45 ). Similar problems have also been reported during cDNA library generation ( 46 ). In addition, sequencing of 5′ LongSAGE clones with poly(A) sequences was not successful (M. Gowda and G.L.…”

Section: Discussionsupporting

confidence: 76%

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

Gowda¹,

Li²,

Alessi

et al. 2006

Nucleic Acids Research

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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 (∼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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Section: Discussionsupporting

confidence: 76%

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

Gowda¹,

Li²,

Alessi

et al. 2006

Nucleic Acids Research

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“…These mismatches can be attributed to the difficulty of determining the precise boundary between each tag in a ditag during data extraction. This finding is due to imprecise cleavage (slippage) by MmeI, a phenomenon that has been observed in other Type IIS restriction endonucleases (13,23,24). Therefore, allowing mismatches in the first 3 and the last 3 nucleotide positions of each tag sequence could significantly increase the 5ЈLS mapping efficiency and specificity to the genome.…”

Section: Resultsmentioning

confidence: 93%

5′ Long serial analysis of gene expression (LongSAGE) and 3′ LongSAGE for transcriptome characterization and genome annotation

Wei

Chiu

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

100

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Complete genome annotation relies on precise identification of transcription units bounded by a transcription initiation site (TIS) and a polyadenylation site (PAS). To facilitate this process, we developed a set of two complementary methods, 5 Long serial analysis of gene expression (LS) and 3LS. These analyses are based on the original SAGE and LS methods coupled with full-length cDNA cloning, and enable the high-throughput extraction of the first and the last 20 bp of each transcript. We demonstrate that the mapping of 5LS and 3LS tags to the genome allows the localization of TIS and PAS. By using 537 tag pairs mapping to the region of known genes, we confirmed that >90% of the tag pairs appropriately assigned to the first and last exons. Moreover, by using tag sequences as primers for RT-PCRs, we were able to recover putative full-length transcripts in 81% of the attempts. This large-scale generation of transcript terminal tags is at least 20 -40 times more efficient than full-length cDNA cloning and sequencing in the identification of complete transcription units. The apparent precision and deep coverage makes 5LS and 3LS an advanced approach for genome annotation through whole-transcriptome characterization.genome annotation ͉ full-length cDNA ͉ transcription analysis T he complete genome sequences of human and other model organisms (1-5) have raised questions as to the accuracy of previously used gene annotation algorithms. Current genome annotations are mostly based on the growing but still limited cDNA sequence databases. Computational gene prediction algorithms, even when trained based on current cDNA data sets, may not provide reliable ab initio predictions of new genes, and all predictions need to be validated by further experimental evidence (6, 7).Efforts in large scale full-length cDNA sequencing (8-11; reviewed in ref. 12) provide not only the complete sequences of expressed genes but also the ability to locate transcription initiation sites (TISs), polyadenylation sites (PASs), as well as splicing junctions. The localization of these functional sites related to transcripts in the context of the genome can greatly help to define the surrounding regulatory elements such as promoters. However, sequencing all possible full-length transcript clones is an expensive and cumbersome process. Despite current progress, complete disclosure of the transcriptome has yet to be achieved. It is clear, therefore, that the precise identification of all genes and their regulatory elements will require more comprehensive and facile technologies with greater throughput to provide the necessary empirical evidence.Serial analysis of gene expression (SAGE) (13, 14) and massively parallel signature sequencing (MPSS) (15) are high-throughput methods that use short tags (14-21 bp of internal transcript signatures) to count transcripts in transcriptomes. When mapped to assembled genome sequences, these short tags help to locate transcription units in the context of the genome. Because concatenation of these short tags and t...

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“…The polyA tail sequences were removed by GsuI digestion (Shibata et al, 2001), followed by phenol chloroform purification and size fractionation of cDNAs using a Sepharose G50 column to collect cDNA fragments greater than 150 bp. Due to the preliminary RNA amplification step, cDNA samples were enriched for short (,500 bp) fragments.…”

Section: Pyrosequencing Of Arna From Laser-captured Samplesmentioning

confidence: 99%

Enlarging Cells Initiating Apomixis inHieracium praealtumTransition to an Embryo Sac Program prior to Entering Mitosis

Okada

Hu²,

Tucker³

et al. 2013

Plant Physiology

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Hieracium praealtum forms seeds asexually by apomixis. During ovule development, sexual reproduction initiates with megaspore mother cell entry into meiosis and formation of a tetrad of haploid megaspores. The sexual pathway ceases when a diploid aposporous initial (AI) cell differentiates, enlarges, and undergoes mitosis, forming an aposporous embryo sac that displaces sexual structures. Embryo and endosperm development in aposporous embryo sacs is fertilization independent. Transcriptional data relating to apomixis initiation in Hieracium spp. ovules is scarce and the functional identity of the AI cell relative to other ovule cell types is unclear. Enlarging AI cells with undivided nuclei, early aposporous embryo sacs containing two to four nuclei, and random groups of sporophytic ovule cells not undergoing these events were collected by laser capture microdissection. Isolated amplified messenger RNA samples were sequenced using the 454 pyrosequencing platform and comparatively analyzed to establish indicative roles of the captured cell types. Transcriptome and protein motif analyses showed that approximately one-half of the assembled contigs identified homologous sequences in Arabidopsis (Arabidopsis thaliana), of which the vast majority were expressed during early Arabidopsis ovule development. The sporophytic ovule cells were enriched in signaling functions. Gene expression indicative of meiosis was notably absent in enlarging AI cells, consistent with subsequent aposporous embryo sac formation without meiosis. The AI cell transcriptome was most similar to the early aposporous embryo sac transcriptome when comparing known functional annotations and both shared expressed genes involved in gametophyte development, suggesting that the enlarging AI cell is already transitioning to an embryo sac program prior to mitotic division.

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Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing

Cited by 12 publications

References 14 publications

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

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

5′ Long serial analysis of gene expression (LongSAGE) and 3′ LongSAGE for transcriptome characterization and genome annotation

Enlarging Cells Initiating Apomixis inHieracium praealtumTransition to an Embryo Sac Program prior to Entering Mitosis

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