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Methods Mol Biol. 2011;700:89-104. doi: 10.1007/978-1-61737-954-3_7.

Bar-coded, multiplexed sequencing of targeted DNA regions using the Illumina Genome Analyzer.

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1
Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA.

Abstract

To date, genome-wide association (GWA) studies, in which thousands of markers throughout the genome are simultaneously genotyped, have identified hundreds of loci underlying disease susceptibility. These regions typically span 5-100 kb, and resequencing efforts to identify potential functional variants within these loci represent the next logical step in the genetic characterization pipeline. Next-generation DNA sequencing technologies are, in principle, well-suited for this task, yet despite the massive sequencing capability afforded by these platforms, the present-day reality is that it remains difficult, time-consuming, and expensive to resequence large numbers of samples across moderately sized genomic regions. To address this obstacle, we developed a generalized framework for multiplexed resequencing of targeted regions of the human genome on the Illumina Genome Analyzer using degenerate, indexed DNA sequence barcodes ligated to fragmented DNA prior to sequencing. Using this method, the DNA of multiple individuals can be simultaneously sequenced at several regions. We find that achieving adequate coverage is one of the most important factors in the design of an experiment, but other key considerations include whether the objective is to discover genetic variants for genotyping later by a separate method, to genotype all identified variants by sequencing, or to exhaustively identify all common and rare variants in the region. Given the massive bandwidth of next-generation sequencing technologies and their low inherent throughput in terms of sequencing arrays per week, multiplexed sequencing using the barcoding approach offers a clear mechanism for focusing bandwidth to a smaller region across many more individuals or samples.

PMID:
21204029
DOI:
10.1007/978-1-61737-954-3_7
[Indexed for MEDLINE]

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