BackgroundDespite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting.ResultsWe leveraged sequence and intensity pattern features responsible for diminished coverage and accuracy and developed a novel algorithm, sPROFILER, which resolved >80% of no-calls from GSEQ and allowed 99.6% (range: 99.2-99.8%) of sequence to be called, while maintaining overall accuracy at >99.8% based upon dideoxy sequencing comparison.ConclusionsTogether, these findings provide insight into critical issues for disease-centered resequencing protocols suitable for clinical application and support the use of array-based resequencing technology as a valuable molecular diagnostic tool for pediatric SNHL and other genetic diseases with substantial genetic heterogeneity.

This unit provides an overview of oligo hybridization–based resequencing and a wide range of considerations for implementing the technology and analyzing the resulting data. The specific technology discussed is the Affymetrix GeneChip CustomSeq Resequencing Array platform. Concepts related to array design, experimental protocols, and base-calling using existing algorithms are presented. Details that should be evaluated during development of sequence tiling, target amplification, and PCR protocols are addressed. An overview of the Affymetrix GeneChip Sequence Analysis Software (GSEQ) is provided, along with factors that influence base-calling coverage and accuracy. Also outlined are performance measures that can be used to characterize base-calling with resequencing arrays, as well as factors known to affect their performance. Limitations associated with detection of insertions and deletions (indels) are discussed, with empirical data from our experiments used to outline possible approaches to indel detection. Critical topics in the design, implementation, and analysis of targeted sequencing arrays not previously discussed in detail are highlighted.