Enrichment of sequencing targets from the human genome by solution hybridization

Tewhey, Ryan; Nakano, Masakazu; Wang, Xiaoyun; Pabón-Peña, Carlos; Novak, Barbara; Giuffre, A.; Lin, Eric Y; Happe, Scott; Roberts, Doug N; LeProust, Emily; Topol, Eric J.; Harismendy, Olivier; Frazer, Kelly A.

doi:10.1186/gb-2009-10-10-r116

Cited by 109 publications

(107 citation statements)

References 34 publications

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“…Some requested target regions were not covered by the NimbleGen and SureSelect designs (9.3% and 8.3%, respectively; Table 2) due to repetitive regions. These percentages are consistent with other reports focused on exon sequence capture (11,12). However, the proportion of protein coding regions included in the SureSelect bait design was comparatively better than that covered by NimbleGen (97.7 and 93.6%, respectively; Table 2), reflecting different methods for defining repetitive regions and complementary oligonucleotide selection.…”

Section: Resultssupporting

confidence: 90%

“…Our sequencing results are consistent with other studies reporting utility of these technologies for diagnosis of other genetic diseases (11,15,23) and suggest that massively parallel sequencing is suitable for genetic testing of NSHL. A key concern for any diagnostic test is sensitivity, as it is critical that pathogenic mutations are not missed.…”

Section: Discussionsupporting

confidence: 91%

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Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

Shearer

DeLuca²,

Hildebrand³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

298

302

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The extreme genetic heterogeneity of nonsyndromic hearing loss (NSHL) makes genetic diagnosis expensive and time consuming using available methods. To assess the feasibility of targetenrichment and massively parallel sequencing technologies to interrogate all exons of all genes implicated in NSHL, we tested nine patients diagnosed with hearing loss. Solid-phase (NimbleGen) or solution-based (SureSelect) sequence capture, followed by 454 or Illumina sequencing, respectively, were compared. Sequencing reads were mapped using GSMAPPER, BFAST, and BOWTIE, and pathogenic variants were identified using a custom-variant calling and annotation pipeline (ASAP) that incorporates publicly available in silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2, and Align-GVGD). Samples included one negative control, three positive controls (one biological replicate), and six unknowns (10 samples total), in which we genotyped 605 single nucleotide polymorphisms (SNPs) by Sanger sequencing to measure sensitivity and specificity for SureSelect-Illumina and NimbleGen-454 methods at saturating sequence coverage. Causative mutations were identified in the positive controls but not in the negative control. In five of six idiopathic hearing loss patients we identified the pathogenic mutation. Massively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss.deafness | genomics | Usher syndrome | diagnostics | next-generation sequencing H ereditary sensorineural hearing loss (SNHL) is the most common sensory impairment in humans (1, 2). In developed countries, two-thirds of prelingual-onset SNHL is estimated to have a genetic etiology, of which ∼70% is nonsyndromic hearing loss (NSHL). Eighty percent of NSHL is autosomal recessive nonsyndromic hearing loss (ARNSHL), ∼20% is autosomal dominant (AD), and the remainder is composed of X-linked and mitochondrial forms (1, 3). To date, 134 deafness loci have been identified, and 32 recessive (DFNB), 23 dominant (DFNA) and 2 X-linked (DFNX) genes have been cloned; 8 genes are associated with both ARNSHL and ADNSHL (4).Establishing a genetic diagnosis of NSHL is a critical component of the clinical evaluation of deaf and hard-of-hearing persons and their families. If a genetic cause of hearing loss is determined, it is possible to provide families with prognostic information, recurrence risks, and improved habilitation options. For persons diagnosed with Usher syndrome, preventative measures including sunlight protection and vitamin therapy can be implemented to minimize the rate of progression of retinitis pigmentosa (5). Most current genetic testing strategies for NSHL rely on a gene-specific Sanger sequencing approach. Because mutations in a single gene, GJB2 (DFNB1), account for up to 50% of ARNSHL in many world populations (6), this approach has changed the evaluation of patients with presumed ARNSHL. However, the mutation frequency in other genes in persons with NSHL in outbred populat...

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Section: Resultssupporting

confidence: 90%

Section: Discussionsupporting

confidence: 91%

Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

Shearer

DeLuca²,

Hildebrand³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

298

302

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“…Genomic DNA was captured by hybridization in solution to custom-designed cRNA oligonucleotide baits (31) following the manufacturer's protocols (Agilent Technologies). BED files of genomic locations of all cRNA oligonucleotide probes are freely available on request to the authors.…”

Section: Methodsmentioning

confidence: 99%

Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing

Walsh¹,

Lee²,

Casadei³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

437

385

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Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, "next-generation" sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer.nherited mutations in BRCA1 and BRCA2 predispose to high risks of breast and ovarian cancer. Lifetime risks of breast cancer are as high as 80% among women with mutations in these genes, and lifetime risks of ovarian cancer are greater than 40% for carriers of BRCA1 mutations and greater than 20% for carriers of BRCA2 mutations (1). Inherited mutations in the Fanconi anemia genes BRIP1 (FANCJ) and PALB2 (FANCN) are associated with 20-50% lifetime risks of breast cancer (2, 3). Inherited mutations in TP53, PTEN, STK11, and CDH1 are associated with moderate to very high risks of breast cancer in the context of Li-Fraumeni syndrome, Cowden syndrome, Peutz-Jeughers syndrome, and hereditary diffuse gastric cancer syndrome, respectively (4, 5, 6, 7). Inherited mutations in several of the genes responsible for hereditary nonpolyposis colon cancer and endometrial cancer are also associated with elevated risks of ovarian cancer (8).Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice for women with severe family histories of breast or ovarian cancer, whether newly diagnosed or still clinically asymptomatic. However, as many as 50% of breast cancer patients with inherited mutations in BRCA1 and BRCA2 do not have close relatives with breast or ovarian cancer because their mutation is paternally inherited, the family is small, and by chance no sisters or paternal au...

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“…Solution-based hybridization methods have been developed to overcome these disadvantages. In the methods, an excess of biotinylated single-stranded RNA [48,49] or DNA oligos [43] is presented as capture probes in solution. A relatively small amount of input DNA-fragment library is required for hybridization reactions in aqueous phase.…”

Section: Targeted Sequencingmentioning

confidence: 99%

Next-generation sequencing in the clinic: Promises and challenges

et al. 2013

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The advent of next generation sequencing (NGS) technologies has revolutionized the field of genomics, enabling fast and cost-effective generation of genome-scale sequence data with exquisite resolution and accuracy. Over the past years, rapid technological advances led by academic institutions and companies have continued to broaden NGS applications from research to the clinic. A recent crop of discoveries have highlighted the medical impact of NGS technologies on Mendelian and complex diseases, particularly cancer. However, the everincreasing pace of NGS adoption presents enormous challenges in terms of data processing, storage, management and interpretation as well as sequencing quality control, which hinder the translation from sequence data into clinical practice. In this review, we first summarize the technical characteristics and performance of current NGS platforms. We further highlight advances in the applications of NGS technologies towards the development of clinical diagnostics and therapeutics. Common issues in NGS workflows are also discussed to guide the selection of NGS platforms and pipelines for specific research purposes.

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Enrichment of sequencing targets from the human genome by solution hybridization

Abstract: A method for target sequence enrichment from the human genome is described. This hybridization-based approach using oligonucleotide probes in solution has excellent sensitivity and accuracy for calling SNPs

Cited by 109 publications

References 34 publications

Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing

Next-generation sequencing in the clinic: Promises and challenges

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