title: De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley.
abstract: BACKGROUND
  De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable.
  
  
  RESULTS
  To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of approximately 50 kb (N80 approximately 31 kb, N90 approximately 21 kb) and a Q40 of 94%. For approximately 80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes.By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies.
  
  
  CONCLUSIONS
  The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany. steuerna@ipk-gatersleben.de
authors.forename: Burkhard
authors.initials: B
authors.lastname: Steuernagel
authors.forename: Stefan
authors.initials: S
authors.lastname: Taudien
authors.forename: Heidrun
authors.initials: H
authors.lastname: Gundlach
authors.forename: Michael
authors.initials: M
authors.lastname: Seidel
authors.forename: Ruvini
authors.initials: R
authors.lastname: Ariyadasa
authors.forename: Daniela
authors.initials: D
authors.lastname: Schulte
authors.forename: Andreas
authors.initials: A
authors.lastname: Petzold
authors.forename: Marius
authors.initials: M
authors.lastname: Felder
authors.forename: Andreas
authors.initials: A
authors.lastname: Graner
authors.forename: Uwe
authors.initials: U
authors.lastname: Scholz
authors.forename: Klaus F X
authors.initials: KF
authors.lastname: Mayer
authors.forename: Matthias
authors.initials: M
authors.lastname: Platzer
authors.forename: Nils
authors.initials: N
authors.lastname: Stein
mesh_terms: D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D001330:Electronic Data Processing; D017343:Genes, Plant / Q000235:genetics*; D023281:Genomics; D001467:Hordeum / Q000235:genetics*; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards