title: Exome sequencing of a multigenerational human pedigree.
abstract: Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.
authors.affiliation: John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
authors.forename: Dale J
authors.initials: DJ
authors.lastname: Hedges
authors.forename: Dale
authors.initials: D
authors.lastname: Hedges
authors.forename: Dan
authors.initials: D
authors.lastname: Burges
authors.forename: Eric
authors.initials: E
authors.lastname: Powell
authors.forename: Cherylyn
authors.initials: C
authors.lastname: Almonte
authors.forename: Jia
authors.initials: J
authors.lastname: Huang
authors.forename: Stuart
authors.initials: S
authors.lastname: Young
authors.forename: Benjamin
authors.initials: B
authors.lastname: Boese
authors.forename: Mike
authors.initials: M
authors.lastname: Schmidt
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Pericak-Vance
authors.forename: Eden
authors.initials: E
authors.lastname: Martin
authors.forename: Xinmin
authors.initials: X
authors.lastname: Zhang
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Harkins
authors.forename: Stephan
authors.initials: S
authors.lastname: Züchner
mesh_terms: D005091:Exons / Q000235:genetics*; D005191:Family Characteristics; D005260:Female; D015894:Genome, Human / Q000235:genetics*; D005838:Genotype; D006801:Humans; D040582:Inheritance Patterns / Q000235:genetics; D008297:Male; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA