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ARTICLEComparison of Commercially Available Target Enrichment Methods forNext-Generation SequencingK. Bodi, 1, * A. G. Perera, 2, * P. S. Adams, 3 D. Bintzler, 4 K. Dewar, 5 D. S. Grove, 6 J. Kieleczawa, 7R. H. Lyons, 8 T. A. Neubert, 9 A. C. Noll, 2 S. Singh, 10 R. Steen, 11 and M. Zianni 121 Tufts University School of Medicine, Boston, Massachusetts 02452, USA; 2 Stowers Institute for Medical Research, Kansas City,Missouri 64110, USA;3 Trudeau Institute, Saranac Lake, New York, 12983; 4 DNA Analysis, LLC, Cincinnati, Ohio 45212;5 McGill University Montreal, QC H31 0G4, Canada; 6 Penn State University, University Park, Pennsylvania 16802; 7 WyzerBiosciences, Cambridge, Massachusetts 02140;8 University of Michigan, Ann Arbor, Michigan 48109; 9 New York UniversitySchool of Medicine, New York, New York 10016;10 University of Minnesota, 100 Church Street, Minneapolis, Minnesota,55455;11 Harvard Medical School, Boston Massachusetts 02115; and 12 The Ohio State University, Columbus, Ohio 43210Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing(NGS) by allowing researchers to focus on their regions of interest. For the 2010–11 DNA SequencingResearch Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies(Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomicenrichment methods. Both companies were provided with the same genomic sample and challenged tocapture identical genomic locations for DNA NGS. The target region totaled 3.5 Mb and included 31individual genes and a 2-Mb contiguous interval. Each company was asked to design its best assay, performthe capture in replicates, and return the captured material to the DSRG-participating laboratories. Sequenc-ing was performed in two different laboratories on Genome Analyzer IIx systems (Illumina, San Diego, CA,USA). Sequencing data were analyzed for sensitivity, specificity, and coverage of the desired regions. Thesuccess of the enrichment was highly dependent on the design of the capture probes. Overall, coveragevariability was higher for the Agilent samples. As variant discovery is the ultimate goal for a typical targetedsequencing project, we compared samples for their ability to sequence single-nucleotide polymorphisms(SNPs) as a test of the ability to capture both chromosomes from the sample. In the targeted regions, wedetected 2546 SNPs with the NimbleGen samples and 2071 with Agilent’s. When limited to the regions thatboth companies included as baits, the number of SNPs was ?1000 for each, with Agilent and NimbleGenfinding a small number of unique SNPs not found by the other.K EY W ORDS : Agilent, NimbleGen, Illumina, targeted captureINTRODUCTIONIn recent years, we have witnessed tremendous advances innext-generation sequencing (NGS) that have dramaticallydriven down the cost of large-scale DNA sequencing.Nonetheless, the cost of completely sequencing large ge-nomes is still significant. 1 Depending on the goal of study,forexample,theassessmentofgenome-wideassociationstudies,whole genome sequencing creates a large amount of additionaldatathatcancomplicatedataprocessingandanalysis.Toalleviatethese problems, the targeted sequencing of the key portions ofgenomes is often being used to identify variations implicated indisease. Whereas many studies are designed to identify geneticvariation of protein-coding genes in a genome-wide manner(whole exome sequencing), there are also applications where afocus on specific genomic intervals or gene sets is required. Forexample,inthepastyearalone,therehavebeenstudiesidentifyingvariantslinkedtoMeckelsyndrome 2 andFanconi’sAnemia. 3 Inclinical settings, where NGS is used to examine specific genepanels,andsamplenumbersarehigh,itismorecost-effectiveandtime-efficient to target, capture, and sequence only the genomicregionsofinterest.Toaddressthisneed,severalcompanieshavedevelopedtargetedenrichmentmethods.The two most commonly used custom-capture ap-proaches are based on hybridization (either on array orin-solution) or on highly multiplexed PCR. In the array-based hybridization method, high-density microarrayscontainingprobescomplementarytotheregionsofinterestA DDRESS CORRESPONDENCE TO : Kip Bodi, Tufts University School ofMedicine, 136 Harrison Ave., Stearns 803, Boston, MA 02111, USA(Phone: 617-636-3763; E-mail: kip.bodi@tufts.edu).*These are shared first authors.doi: 10.7171/jbt.13-2402-002xxxxxxxxxxxxJournal of Biomolecular Techniques 24:73–86 © 2013 ABRF