Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing

Gnirke, Andreas; Melnikov, Alexandre; Maguire, Jared; Rogov, Peter; LeProust, Emily; Brockman, William W.; Fennell, Timothy; Giannoukos, Georgia; Fisher, Sheila; Russ, Carsten; Gabriel, Stacey; Jaffe, David B.; Lander, Eric S.; Nusbaum, Chad

doi:10.1038/nbt.1523

Cited by 1,281 publications

(1,184 citation statements)

References 25 publications

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“…Library Construction was performed as described previously, using 50-200 ng of DNA sheared by sonication to B100-400 bp before end-repair, dA addition and ligation of indexed, Illumina sequencing adaptors. 33 Enrichment of target sequences (3320 exons of 182 cancer-related genes and 37 introns from 14 genes recurrently rearranged in cancer representing approximately 1.1 Mb of the human genome) was achieved by solution-based hybrid capture with a custom Agilent SureSelect biotinylated RNA baitset. 33 The selected libraries were sequenced on an Illumina HiSeq 2000 platform using 49 Â 49 paired-end reads.…”

Section: Methodsmentioning

confidence: 99%

“…33 Enrichment of target sequences (3320 exons of 182 cancer-related genes and 37 introns from 14 genes recurrently rearranged in cancer representing approximately 1.1 Mb of the human genome) was achieved by solution-based hybrid capture with a custom Agilent SureSelect biotinylated RNA baitset. 33 The selected libraries were sequenced on an Illumina HiSeq 2000 platform using 49 Â 49 paired-end reads. Sequence data from genomic DNA was mapped to the reference human genome (hg19) using the Burrows-Wheeler Aligner and were processed using the publicly available SAMtools, Picard and Genome Analysis Toolkit.…”

Section: Methodsmentioning

confidence: 99%

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Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Wang²,

et al. 2014

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Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 lm of formalin-fixed, paraffinembedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 Â and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0-10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0-7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Wang²,

et al. 2014

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“…11 The manufacturer's protocol version 1.0, compatible with Illumina paired-end sequencing, was used with the exception that DNA fragment size and quality were measured with 2% agarose gels stained with Sybr Gold. Captured regions totaled B38 Mb.…”

Section: Exome Analysismentioning

confidence: 99%

Exome sequencing and SNP analysis detect novel compound heterozygosity in fatty acid hydroxylase-associated neurodegeneration

et al. 2011

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Fatty acid hydroxylase-associated neurodegeneration due to fatty acid 2-hydroxylase deficiency presents with a wide range of phenotypes including spastic paraplegia, leukodystrophy, and/or brain iron deposition. All previously described families with this disorder were consanguineous, with homozygous mutations in the probands. We describe a 10-year-old male, from a non-consanguineous family, with progressive spastic paraplegia, dystonia, ataxia, and cognitive decline associated with a sural axonal neuropathy. The use of high-throughput sequencing techniques combined with SNP array analyses revealed a novel paternally derived missense mutation and an overlapping novel maternally derived B28-kb genomic deletion in FA2H. This patient provides further insight into the consistent features of this disorder and expands our understanding of its phenotypic presentation. The presence of a sural nerve axonal neuropathy had not been previously associated with this disorder and so may extend the phenotype.

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“…Samples were prepared using the SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA) (Gnirke et al 2009). Briefly, each DNA sample was sheared, bluntend repaired, and adaptor-ligated using indexed adapters.…”

Section: Methodsmentioning

confidence: 99%

Diagnostic Exome Sequencing and Tailored Bioinformatics of the Parents of a Deceased Child with Cobalamin Deficiency Suggests Digenic Inheritance of the MTR and LMBRD1 Genes

González

et al. 2014

JIMD Reports

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Disorders of cobalamin deficiency are a heterogeneous group of disorders with at least 19 autosomal recessive-associated genes. Familial samples of an infant who died due to presumed cobalamin deficiency were referred for clinical exome sequencing. The patient died before obtaining a blood sample or skin biopsy, autopsy was declined, and DNA yielded from the newborn screening blood spot was insufficient for diagnostic testing. Whole-exome sequencing of the mother, father, and unaffected sister and tailored bioinformatics analysis was applied to search for mutations in underlying disorders with recessive inheritance. This approach identified alterations within two genes, each of which was carried by one parent. The mother carried a missense alteration in the MTR gene (c.3518C>T; p.P1173L) which was absent in the father and the sister. The father carried a translational frameshift alteration in the LMBRD1 gene (c.1056delG; p.L352Lfs*18) which was absent in the mother and present in the heterozygous state in the sister. These mutations in the MTR (MIM# 156570) and LMBRD1 (MIM# 612625) genes have been described in patients with disorders of cobalamin metabolism complementation groups cblG and cblF, respectively. The child's clinical presentation and biochemical results demonstrated overlap with both cblG and cblF. Sanger sequencing using DNA from the infant's blood spot confirmed the inheritance of the two alterations in compound heterozygous form. We present the first example of exome sequencing leading to a diagnosis in the absence of the affected patient. Furthermore, the data support the possibility for potential digenic inheritance associated with cobalamin deficiency.

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Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing

Cited by 1,281 publications

References 25 publications

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Exome sequencing and SNP analysis detect novel compound heterozygosity in fatty acid hydroxylase-associated neurodegeneration

Diagnostic Exome Sequencing and Tailored Bioinformatics of the Parents of a Deceased Child with Cobalamin Deficiency Suggests Digenic Inheritance of the MTR and LMBRD1 Genes

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