title: Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations.
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.
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, The Netherlands.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Harakalova
authors.forename: Isaäc J
authors.initials: IJ
authors.lastname: Nijman
authors.forename: Jelena
authors.initials: J
authors.lastname: Medic
authors.forename: Michal
authors.initials: M
authors.lastname: Mokry
authors.forename: Ivo
authors.initials: I
authors.lastname: Renkens
authors.forename: Jan D
authors.initials: JD
authors.lastname: Blankensteijn
authors.forename: Wigard
authors.initials: W
authors.lastname: Kloosterman
authors.forename: Annette F
authors.initials: AF
authors.lastname: Baas
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
mesh_terms: D000368:Aged; D017544:Aortic Aneurysm, Abdominal / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D001019:Aortic Rupture / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D002899:Chromosomes, Human, Pair 9; D058893:DNA Barcoding, Taxonomic; D004252:DNA Mutational Analysis; D005189:False Positive Reactions; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D005787:Gene Frequency; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D009154:Mutation; D009426:Netherlands; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D010865:Pilot Projects; D011379:Prognosis; D015203:Reproducibility of Results