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.
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References
International HapMap Consortium (2007) A second generation human haplotype map of over 3.1 million SNPs. Nature 449:851–861
Wellcome Trust Case Control Consortium (2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447:661–678
Zondervan KT, Cardon LR (2007) Designing candidate gene and genome-wide case-control association studies. Nat Protoc 2:2492–2501
Milosavljevic A et al (2005) Pooled genomic indexing of rhesus macaque. Genome Res 15:292–301
Parameswaran P, Jalili R, Tao L, Shokralla S, Gharizadeh B, Ronaghi M, Fire AZ (2007) A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res 35:e130
Meyer M, Stenzel U, Myles S, Prufer K, Hofreiter M (2007) Targeted high-throughput sequencing of tagged nucleic acid samples. Nucleic Acids Res 35:e97
Hamady M, Walker JJ, Harris JK, Gold NJ, Knight R (2008) Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods 5:235–237
Paired-end sequencing sample preparation guide. Illumina, Inc. Part# 1005063 Rev. B
Albert TJ et al (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods 4(11):891–892
Porreca GJ et al (2007) Multiplex amplification of large sets of human exons. Nat Methods 4(11):931–936
Gnirke A et al (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel, targeted sequencing. Nat Biotechnol 27(2):182–189
Ng SB et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272–276
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Szelinger, S., Kurdoglu, A., Craig, D.W. (2011). Bar-Coded, Multiplexed Sequencing of Targeted DNA Regions Using the Illumina Genome Analyzer. In: DiStefano, J. (eds) Disease Gene Identification. Methods in Molecular Biology, vol 700. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61737-954-3_7
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DOI: https://doi.org/10.1007/978-1-61737-954-3_7
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61737-954-3
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