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

BMC Genomics
Burkhard SteuernagelNils Stein

Abstract

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 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 t...Continue Reading

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Citations

Mar 16, 2013·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·Chiara BiselliGiampiero Valè
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Datasets Mentioned

BETA
SRP001149
37963
MG1655
U00096

Methods Mentioned

BETA
random shearing
454 sequencing
clone-by-clone
Illumina sequencing
454
DNA assay

Software Mentioned

R
MIRA assemblis
GenomeThreader
BlastN
Rocks4
MIRA
Linux
GS FLX
Newbler
SSAHA

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