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Low-pass shotgun sequencing of barley


Low-pass shotgun sequencing of the barley genome facilitates rapid identification of genes, conserved non-coding sequences and novel repeats


Thomas Wicker1, Apurva Narechania2, Francois Sabot3, Joshua Stein2, Giang Thi Ha Vu5,6, Andreas Graner5, Doreen Ware2,4, Nils Stein5,


1 Institute of Plant Biology, University Zurich, Zollikerstrasse 107, CH-8008 Zurich

2 Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724

3 Laboratoire Génome et Développement des Plantes, UMR 5096 CNRS-IRD-Université de Perpignan, 52 Avenue Paul Alduy, F-66860 Perpignan, France

4 4United States Department of Agriculture–Agricultural Research Service (USDA–ARS) North Atlantic Area (NAA) Plant, Soil & Nutrition Laboratory Research Unit, Ithaca, New York 15853 USA

5 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben

6 current adress: Institute of Biological, Environmental and Rural Sciences (IBERS), Edward Llwyd Buiding, Aberystwyth University, Ceredigion, SY23 3DA UK

Corresponding Author

Dr. Nils Stein
Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK)
Genebank Department
Corrensstr. 3
D-06466 Gatersleben
Tel.: +49-39482-5522
Fax.: +49-39482-5595
email: stein@ipk-gatersleben.de


Background: Barley has one of the largest and most complex genomes of all economically important food crops. The rise of new short read sequencing technologies such as Illumina/Solexa permits such large genomes to be effectively sampled at relatively low cost. Based on the corresponding sequence reads a Mathematically Defined Repeat (MDR) index can be generated to map repetitive regions in genomic sequences.
Results: We have generated 574 Mbp of Illumina/Solexa sequences from barley total genomic DNA, representing about 10% of a genome equivalent. From these sequences we generated an MDR index which was then used to identify and mark repetitive regions in the barley genome. Comparison of the MDR plots with expert repeat annotation drawing on the information already available for known repetitive elements revealed a significant correspondence between the two methods. MDR-based annotation allowed for the identification of dozens of novel repeat sequences, though, which were not recognised by hand-annotation. The MDR data was also used to identify gene-containing regions by masking of repetitive sequences in eight de-novo sequenced bacterial artificial chromosome (BAC) clones. For half of the identified candidate gene islands indeed gene sequences could be identified. MDR data were only of limited use, when mapped on genomic sequences from the closely related species Triticum monococcum as only a fraction of the repetitive sequences was recognised.
Conclusion: An MDR index for barley, which was obtained by whole-genome Illumina/Solexa sequencing, proved as efficient in repeat identification as manual expert annotation . Circumventing the labour-intensive step of producing a specific repeat library for expert annotation, an MDR index provides an elegant and efficient resource for the identification of repetitive and low-copy (i.e potentially gene-containing sequences) regions in uncharacterised genomic sequences. The restriction that a particular MDR index can not be used across species is outweighed by the low costs of Illumina/Solexa sequencing which makes any chosen genome accessible for whole-genome sequence sampling.