Open Access Highly Accessed Methodology article

De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley

Burkhard Steuernagel1, Stefan Taudien2, Heidrun Gundlach3, Michael Seidel3, Ruvini Ariyadasa1, Daniela Schulte1, Andreas Petzold2, Marius Felder2, Andreas Graner1, Uwe Scholz1, Klaus FX Mayer3, Matthias Platzer2 and Nils Stein1*

Author Affiliations

1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Correnstr. 3, D-06466 Gatersleben, Germany

2 Leibniz Institute for Age Research, Fritz Lipmann Institute (FLI), Beutenbergstr. 11, D-07745 Jena, Germany

3 MIPS/IBIS, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany

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BMC Genomics 2009, 10:547  doi:10.1186/1471-2164-10-547

Published: 20 November 2009



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.


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 ~50 kb (N80 ~31 kb, N90 ~21 kb) and a Q40 of 94%. For ~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.


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.