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Nat Biotechnol. 2014 Oct;32(10):1019-25. doi: 10.1038/nbt.2959. Epub 2014 Aug 17.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Author information

1
1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2].
2
1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2] Cergentis B.V., Utrecht, the Netherlands. [3].
3
Cergentis B.V., Utrecht, the Netherlands.
4
Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
5
Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands.
6
Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands.
7
1] Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands. [2] Department of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands.
8
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
9
Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
10
Leiden Genome Technology Center, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands.
11
1] Department of Medical Protein Research, VIB, Ghent, Belgium. [2] Department of Biochemistry, Ghent University, Ghent, Belgium.
12
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands.
13
Division of Molecular Pathology and Cancer Genomics Center, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
14
Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands.
15
Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands.
16
1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2] Cergentis B.V., Utrecht, the Netherlands.

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.

PMID:
25129690
DOI:
10.1038/nbt.2959
[Indexed for MEDLINE]
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