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

ABSTRACT: 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 approximat

SUBMITTER: Takasuga A 

PROVIDER: S-EPMC92572 | biostudies-literature | 2001 Nov

REPOSITORIES: biostudies-literature