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Nucleic Acids Res. 2014 Jun;42(10):e82. doi: 10.1093/nar/gku218. Epub 2014 Mar 20.

Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection.

Author information

1
Department of Orthopaedic Surgery, Washington University School of Medicine, 660 S Euclid Ave., St Louis, MO 63110, USA.
2
Department of Pediatrics, Washington University School of Medicine, 660 S Euclid Ave., St Louis, MO 63110, USA Department of Genetics, Washington University School of Medicine, 660 S Euclid Ave., St Louis, MO 63110, USA.
3
Genome Technology and Systems Biology, NHLI, Imperial College, London, UK.
4
Department of Orthopaedic Surgery, Washington University School of Medicine, 660 S Euclid Ave., St Louis, MO 63110, USA Department of Pediatrics, Washington University School of Medicine, 660 S Euclid Ave., St Louis, MO 63110, USA Department of Neurology, Washington University School of Medicine, 660 S Euclid Ave., St Louis, MO 63110, USA gurnettc@neuro.wustl.edu.

Abstract

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest.

PMID:
24682816
PMCID:
PMC4041413
DOI:
10.1093/nar/gku218
[Indexed for MEDLINE]
Free PMC Article
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