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Nucleic Acids Research Advance Access published online on August 27, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn502
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

Richard Cronn1,*, Aaron Liston2,3, Matthew Parks2, David S. Gernandt4, Rongkun Shen2,3 and Todd Mockler2,3

1Pacific Northwest Research Station, USDA Forest Service, Corvallis, OR 97331, 2Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, 3Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA and 4Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, México DF 04510, Mexico

*To whom correspondence should be addressed. Tel: +541 750 7291; Fax: +541 750 7329; Email: rcronn{at}fs.fed.us

Received April 9, 2008. Revised June 26, 2008. Accepted July 21, 2008.

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified ~120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligated to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate references. Assemblies for these eight genomes are estimated at 88–94% complete, with an average sequence depth of 55x to 186x. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity.

Present address: Rongkun Shen, Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA


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