Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

Autor: Wigard P. Kloosterman, Magdalena Harakalova, Annette F. Baas, Jelena Medic, Isaac J. Nijman, Michal Mokry, Ivo Renkens, Jan D. Blankensteijn, Edwin Cuppen
Přispěvatelé: Hubrecht Institute for Developmental Biology and Stem Cell Research, Surgery, ICaR - Ischemia and repair
Rok vydání: 2011
Předmět:
Male
Heterozygote
Candidate gene
Aortic Rupture
DNA Mutational Analysis
Common disease
Pooling
Targeted genomic enrichment
Pharmaceutical Science
Pilot Projects
Locus (genetics)
Biology
Article
Deep sequencing
DNA sequencing
Gene Frequency
Genetics
DNA Barcoding
Taxonomic
Humans
Genetics(clinical)
False Positive Reactions
Genetic Predisposition to Disease
Allele frequency
Genetic Association Studies
Genetics (clinical)
Aged
Netherlands
Oligonucleotide Array Sequence Analysis
SOLiD next-generation sequencing
Gene Expression Profiling
High-Throughput Nucleotide Sequencing
Reproducibility of Results
Rare variants
Prognosis
genomic DNA
Phenotype
Genomic DNA pooling
Mutation
Abdominal aortic aneurysm
Molecular Medicine
Female
DNA microarray
Chromosomes
Human
Pair 9
Cardiology and Cardiovascular Medicine
Aortic Aneurysm
Abdominal
Zdroj: Journal of Cardiovascular Translational Research, 4(3), 271-280. Springer New York
Harakalova, M, Nijman, I J, Medic, J, Mokry, M, Renkens, I, Blankensteijn, J D, Kloosterman, W, Baas, A F & 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, vol. 4, no. 3, pp. 271-280 . https://doi.org/10.1007/s12265-011-9263-5
Journal of Cardiovascular Translational Research
ISSN: 1937-5395
1937-5387
DOI: 10.1007/s12265-011-9263-5
Popis: 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.
Databáze: OpenAIRE
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