Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

TY - JOUR

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

SN - 1088-9051

VL - 14

SP - 665

EP - 671

JO - Genome Research

JF - Genome Research

IS - 4

ER -