Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36,000 bovine ESTs.

We determined 36,310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3'-ends. This permitted high quality reading of sequences from the 3'-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16,358 non-redundant ESTs were derived from approximately 6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.

[1]  M. Georges,et al.  Mapping quantitative trait loci controlling milk production in dairy cattle by exploiting progeny testing. , 1995, Genetics.

[2]  Timothy B. Stockwell,et al.  The Sequence of the Human Genome , 2001, Science.

[3]  T. F. Laughlin,et al.  Comparative anchor tagged sequences (CATS) for integrative mapping of mammalian genomes , 1997, Nature Genetics.

[4]  D. Mccormick.  Sequence the Human Genome , 1986, Bio/Technology.

[5]  Pascal Leroy,et al.  An imprinted QTL with major effect on muscle mass and fat deposition maps to the IGF2 locus in pigs , 1999, Nature Genetics.

[6]  P. Lijnzaad,et al.  A physical map of 30,000 human genes. , 1998, Science.

[7]  Michel Georges,et al.  A deletion in the bovine myostatin gene causes the double–muscled phenotype in cattle , 1997, Nature Genetics.

[8]  L. Andersson,et al.  Genetic mapping of quantitative trait loci for growth and fatness in pigs. , 1994, Science.

[9]  H. Aso,et al.  A preadipocyte clonal line from bovine intramuscular adipose tissue: nonexpression of GLUT-4 protein during adipocyte differentiation. , 1995, Biochemical and biophysical research communications.

[10]  M. Georges,et al.  A QTL with major effect on milk yield and composition maps to bovine Chromosome 14 , 1998, Mammalian Genome.

[11]  Leif Andersson,et al.  A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus , 1999, Nature Genetics.

[12]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[13]  M. Rebeiz,et al.  An ordered comparative map of the cattle and human genomes. , 2000, Genome research.

[14]  G. L. Bennett,et al.  Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle. , 2001, Genome research.

[15]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[16]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[17]  K Shimizu,et al.  BodyMap: a collection of 3' ESTs for analysis of human gene expression information. , 2000, Genome research.