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Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations

Harakalova, M. and Nijman, I.J. and Medic, J. and Mokry, M. and Renkens, I. and Blankensteijn, J. D. and Kloosterman, W.P. and Baas, A. F. and Cuppen, E. (2011) Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations. Journal of Cardiovascular Translational Research, 4, 271-80. ISSN 1937-5395.

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Official URL: http://dx.doi.org/10.1007/s12265-011-9263-5

Abstract

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy.

Item Type:Article
Institutes:Hubrecht Instituut
ID Code:12616
Deposited On:07 Sep 2012 14:14
Last Modified:19 Sep 2012 16:37

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