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Open Access Highly Accessed Research article

Sequencing of BAC pools by different next generation sequencing platforms and strategies

Stefan Taudien1*, Burkhard Steuernagel2, Ruvini Ariyadasa2, Daniela Schulte2, Thomas Schmutzer2, Marco Groth1, Marius Felder1, Andreas Petzold1, Uwe Scholz2, Klaus FX Mayer3, Nils Stein2 and Matthias Platzer1

Author Affiliations

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

2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, 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 Research Notes 2011, 4:411  doi:10.1186/1756-0500-4-411

Published: 14 October 2011

Abstract

Background

Next generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs) improve the assemblies by scaffolding and whether barcoding of BACs is dispensable.

Results

Sequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library.

Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%.

Conclusion

Optimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.

Keywords:
BAC pools; next generation sequencing; 454; Illumina; barcoding; mate pairs; scaffolding; barley