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.
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Acknowledgements
We thank T.P. Driessen for figure graphics. This work was supported by the Netherlands Genomics Initiative (NGI) pre-seed grants 93608003 and 93611010 and a proof of concept grant from the Cancer Genomics Center (CGC) to W.d.L. and an Innovation Credit from NL Agency to Cergentis. P.J.P.d.V. is supported by a Dutch Cancer Foundation grant KWF (2009-4459 to J.A.F., J.W.M. and W.d.L.).
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P.J.P.d.V., E.d.W., M.v.M., E.S. and W.d.L. conceived the experiments and analyzed the data; P.J.P.d.V., M.Y., M.v.d.H., P.K., M.J.A.M.V., Y.W., H.T., P.H.L.K., G.G. and E.S. performed the experiments and analyzed the data; M.v.M. and W.d.L. invented TLA. All other authors provided patient samples and analyzed data. E.S., E.d.W. and W.d.L. wrote the manuscript with input from P.J.P.d.V., P.H.L.K., G.G., D.S., B.Y., J.J., L.J.C.M.v.Z., M.L., M.C., B.S.-R., K.W.v.D., M.J.L.L., H.K.P.v.A., J.T.d.D., J.W.M. and M.v.M.
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P.J.P.d.V., M.J.A.M.V., P.H.L.K. and G.G. are shareholders of Cergentis. E.d.W. is co-founder and shareholder of Cergentis and works part time for Cergentis. W.d.L. is co-founder and shareholder of Cergentis. Based on a consultancy agreement between the Hubrecht Institute and Cergentis, W.d.L. works one day per week for Cergentis.
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de Vree, P., de Wit, E., Yilmaz, M. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32, 1019–1025 (2014). https://doi.org/10.1038/nbt.2959
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DOI: https://doi.org/10.1038/nbt.2959
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