Comparison of Commercially Available Target Enrichment Methods for Next-Generation Sequencing

Bodi, Kip; Perera, Anoja; Adams, Pamela S.; Bintzler, D.; Dewar, Ken; Grove, David; Kieleczawa, Jan; Lyons, Robert H.; Neubert, Thomas A.; Noll, Aaron; Singh, Sanjay; Steen, Robert; Zianni, Michael

doi:10.7171/jbt.13-2402-002

Cited by 108 publications

(85 citation statements)

References 8 publications

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“…Exome sequencing performance metrics (Table 1) demonstrated that the hybridisation capture efficiencies, as determined by the percentage of sequence reads mapping to each exome, were comparable to those reported elsewhere. [8] To determine the proportion of target nucleotides that were sufficiently well sequenced to exclude the presence of non-reference nucleotides, read-depth analysis was performed. For two of the three sequenced individuals, >98% of target bases had a read depth that was ≥30×, which is a conservative read-depth metric that has been widely adopted by the diagnostic sequencing community [9] (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

et al. 2015

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Background. Beukes hip dysplasia (BHD) is an autosomal dominant disorder of variable penetrance that was originally identified in a large South African family of European origin. BHD is characterised by bilateral dysmorphism of the proximal femur, which results in severe degenerative osteoarthropathy. Previous studies mapped the disorder to a 3.34 Mb region on chromosome 4q35. Objective. To fine-map the BHD locus and identify the disease-causing mutation by direct sequencing. Results. The linked BHD allele was refined to 1.33 Mb, reducing the number of candidate genes from 25 to 16. Analysis of protein coding and invariant splice-site sequences in three distantly related individuals identified a single-candidate disease-causing variant c.868T>C within exon 8 of the ubiquitin-fold modifier 1 (Ufm1)-specific peptidase 2 gene, UFSP2. The presence of this unique mutation was confirmed in all 17 affected members of the BHD family who were genotyped. The mutation segregated with the BHD phenotype in the extended family with a two-point (single marker) LOD score of 10.4 (θ = 0.0 and 80% penetrance). The mutation predicts the substitution of a highly conserved amino acid, p.Tyr290His, in the encoded protein. In vitro functional assays performed using purified recombinant wild-type and mutant UFSP2 protein demonstrated that the BHD mutation abolishes UFSP2-mediated C-terminal cleavage of its substrate, Ufm1. Conclusion. We report a unique UFSP2 mutation that segregates with the BHD phenotype. The predicted amino acid substitution inactivates UFSP2 proteolytic function, thus implicating the ubiquitin-fold modifier 1 cascade in this form of severe hip osteoarthropathy. The facile polymerase chain reaction-based assay we describe could be used to confirm the diagnosis of BHD, or for presymptomatic testing of members of the extended BHD family.

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Section: Resultsmentioning

confidence: 99%

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

et al. 2015

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“…These regions may include all exons (whole exome sequencing [WES]) and be sequenced to moderate depth, or they may comprise a much smaller fraction of the genome and be sequenced to high depth. 3 Capture probes targeting regions that have been widely studied and implicated in a group of rare heritable disorders can turn HTS into a valuable tool for their affordable diagnosis.…”

Section: Introductionmentioning

confidence: 99%

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Simeoni

Stephens

et al. 2016

Blood

162

187

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“…Targeted analysis of genomic regions longer than 200 kilo-base pairs (bp) is extremely complicated, and therefore targeting schemes usually focus on exons and neglect the introns and regulatory regions (5,6).…”

Section: Cc-by-nc-nd 40 International License Not Peer-reviewed) Is mentioning

confidence: 99%

Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

Gabrieli

Sharim

Michaeli

et al. 2017

Preprint

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Variations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analysis is unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, singlemolecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

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Comparison of Commercially Available Target Enrichment Methods for Next-Generation Sequencing

Cited by 108 publications

References 8 publications

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

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