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Plant Methods. 2012 Mar 30;8:12. doi: 10.1186/1746-4811-8-12.

Targeted parallel sequencing of large genetically-defined genomic regions for identifying mutations in Arabidopsis.

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1
Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA. Khliu@molbio.mgh.harvard.edu.

Abstract

Large-scale genetic screens in Arabidopsis are a powerful approach for molecular dissection of complex signaling networks. However, map-based cloning can be time-consuming or even hampered due to low chromosomal recombination. Current strategies using next generation sequencing for molecular identification of mutations require whole genome sequencing and advanced computational devises and skills, which are not readily accessible or affordable to every laboratory. We have developed a streamlined method using parallel massive sequencing for mutant identification in which only targeted regions are sequenced. This targeted parallel sequencing (TPSeq) method is more cost-effective, straightforward enough to be easily done without specialized bioinformatics expertise, and reliable for identifying multiple mutations simultaneously. Here, we demonstrate its use by identifying three novel nitrate-signaling mutants in Arabidopsis.

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