title: Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure.
abstract: BACKGROUND
  Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.
  
  
  RESULTS
  We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.
  
  
  CONCLUSIONS
  We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
authors.affiliation: J. Craig Venter Institute (formerly The Institute for Genomic Research), 9704 Medical Center Dr., Rockville, MD, USA. rcoyne@jcvi.org
authors.forename: Robert S
authors.initials: RS
authors.lastname: Coyne
authors.forename: Mathangi
authors.initials: M
authors.lastname: Thiagarajan
authors.forename: Kristie M
authors.initials: KM
authors.lastname: Jones
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Wortman
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Tallon
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Haas
authors.forename: Donna M
authors.initials: DM
authors.lastname: Cassidy-Hanley
authors.forename: Emily A
authors.initials: EA
authors.lastname: Wiley
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Smith
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Lee
authors.forename: Mary T
authors.initials: MT
authors.lastname: Couvillion
authors.forename: Yifan
authors.initials: Y
authors.lastname: Liu
authors.forename: Jyoti
authors.initials: J
authors.lastname: Garg
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Pearlman
authors.forename: Eileen P
authors.initials: EP
authors.lastname: Hamilton
authors.forename: Eduardo
authors.initials: E
authors.lastname: Orias
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Eisen
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Methé
mesh_terms: D000818:Animals; D020224:Expressed Sequence Tags; D017125:Genes, Protozoan; D018503:Genome, Protozoan; D048648:Macronucleus; D048631:Micronucleus, Germline; D009693:Nucleic Acid Hybridization; D016808:Tetrahymena thermophila / Q000235:genetics*