title: Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.
abstract: Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.
authors.affiliation: 1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2].
authors.forename: Paula J P
authors.initials: PJ
authors.lastname: de Vree
authors.affiliation: 1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2] Cergentis B.V., Utrecht, the Netherlands. [3].
authors.forename: Elzo
authors.initials: E
authors.lastname: de Wit
authors.affiliation: Cergentis B.V., Utrecht, the Netherlands.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Yilmaz
authors.affiliation: Cergentis B.V., Utrecht, the Netherlands.
authors.forename: Monique
authors.initials: M
authors.lastname: van de Heijning
authors.affiliation: Cergentis B.V., Utrecht, the Netherlands.
authors.forename: Petra
authors.initials: P
authors.lastname: Klous
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Marjon J A M
authors.initials: MJ
authors.lastname: Verstegen
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wan
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Teunissen
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Peter H L
authors.initials: PH
authors.lastname: Krijger
authors.affiliation: Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Geert
authors.initials: G
authors.lastname: Geeven
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Eijk
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Daoud
authors.initials: D
authors.lastname: Sie
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands.
authors.forename: Bauke
authors.initials: B
authors.lastname: Ylstra
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Lorette O M
authors.initials: LO
authors.lastname: Hulsman
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Marieke F
authors.initials: MF
authors.lastname: van Dooren
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Laura J C M
authors.initials: LJ
authors.lastname: van Zutven
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands.
authors.forename: Ans
authors.initials: A
authors.lastname: van den Ouweland
authors.affiliation: 1] Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands. [2] Department of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Sjef
authors.initials: S
authors.lastname: Verbeek
authors.affiliation: 1] Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands. [2] Department of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Ko Willems
authors.initials: KW
authors.lastname: van Dijk
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Marion
authors.initials: M
authors.lastname: Cornelissen
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Atze T
authors.initials: AT
authors.lastname: Das
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
authors.forename: Birgit
authors.initials: B
authors.lastname: Sikkema-Raddatz
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
authors.forename: Eva
authors.initials: E
authors.lastname: van den Berg
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
authors.forename: Pieter
authors.initials: P
authors.lastname: van der Vlies
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
authors.forename: Desiree
authors.initials: D
authors.lastname: Weening
authors.affiliation: Leiden Genome Technology Center, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Johan T
authors.initials: JT
authors.lastname: den Dunnen
authors.affiliation: 1] Department of Medical Protein Research, VIB, Ghent, Belgium. [2] Department of Biochemistry, Ghent University, Ghent, Belgium.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Matusiak
authors.affiliation: 1] Department of Medical Protein Research, VIB, Ghent, Belgium. [2] Department of Biochemistry, Ghent University, Ghent, Belgium.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Lamkanfi
authors.affiliation: Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands.
authors.forename: Marjolijn J L
authors.initials: MJ
authors.lastname: Ligtenberg
authors.affiliation: Division of Molecular Pathology and Cancer Genomics Center, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Petra
authors.initials: P
authors.lastname: ter Brugge
authors.affiliation: Division of Molecular Pathology and Cancer Genomics Center, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Jos
authors.initials: J
authors.lastname: Jonkers
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands.
authors.forename: John A
authors.initials: JA
authors.lastname: Foekens
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands.
authors.forename: John W
authors.initials: JW
authors.lastname: Martens
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Rob
authors.initials: R
authors.lastname: van der Luijt
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands.
authors.forename: Hans Kristian Ploos
authors.initials: HK
authors.lastname: van Amstel
authors.affiliation: Cergentis B.V., Utrecht, the Netherlands.
authors.forename: Max
authors.initials: M
authors.lastname: van Min
authors.affiliation: Cergentis B.V., Utrecht, the Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Splinter
authors.affiliation: 1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2] Cergentis B.V., Utrecht, the Netherlands.
authors.forename: Wouter
authors.initials: W
authors.lastname: de Laat
mesh_terms: D050939:Gene Fusion / Q000235:genetics; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D056426:Genetic Loci / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D009369:Neoplasms / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*