Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing
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Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing. / Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan; Gibbs, Richard A; Brent, Michael R.
In: Genome Research, Vol. 14, No. 4, 2004, p. 665-71.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing
AU - Wu, Jia Qian
AU - Shteynberg, David
AU - Arumugam, Manimozhiyan
AU - Gibbs, Richard A
AU - Brent, Michael R
PY - 2004
Y1 - 2004
N2 - The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially or completely missed by methods based on protein-to-genome mapping. Using primers in exons flanking a single predicted intron, we were able to verify the existence of 59% of these predicted genes. We then attempted to amplify the complete predicted open reading frames of 136 genes that were verified in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.
AB - The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially or completely missed by methods based on protein-to-genome mapping. Using primers in exons flanking a single predicted intron, we were able to verify the existence of 59% of these predicted genes. We then attempted to amplify the complete predicted open reading frames of 136 genes that were verified in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.
KW - Animals
KW - Computational Biology
KW - Genes
KW - Humans
KW - Introns
KW - Open Reading Frames
KW - Predictive Value of Tests
KW - Rats
KW - Reverse Transcriptase Polymerase Chain Reaction
KW - Sequence Analysis, DNA
KW - Software
KW - Untranslated Regions
U2 - 10.1101/gr.1959604
DO - 10.1101/gr.1959604
M3 - Journal article
C2 - 15060008
VL - 14
SP - 665
EP - 671
JO - Genome Research
JF - Genome Research
SN - 1088-9051
IS - 4
ER -
ID: 43976150