Abstract
Despite developments in targeted and whole-genome sequencing, 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. Targeted locus amplification (TLA) is a cross-linking-based technique that generates complex DNA libraries covering >100 kb of contiguous sequence surrounding one primer pair complementary to a short locus-specific sequence. In combination with next-generation sequencing, TLA enables the complete sequencing and haplotyping of targeted regions of interest. Here we outline the basis of TLA, together with a detailed protocol of the technique.
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References
de Vree PJ, de Wit E, Yilmaz M et al (2014) Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32:1019–1025
Snyder MW, Adey A, Kitzman JO et al (2015) Haplotype-resolved genome sequencing: experimental methods and applications. Nat Rev Genet 16:344–358
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760
Nielsen R, Paul JS, Albrechtsen A et al (2011) Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet 12:443–451
DePristo MA, Banks E, Poplin R et al (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491–498
Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079
Acknowledgements
We thank Marieke Simonis for data analysis.
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Hottentot, Q.P., van Min, M., Splinter, E., White, S.J. (2017). Targeted Locus Amplification and Next-Generation Sequencing. In: White, S., Cantsilieris, S. (eds) Genotyping. Methods in Molecular Biology, vol 1492. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6442-0_13
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DOI: https://doi.org/10.1007/978-1-4939-6442-0_13
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6440-6
Online ISBN: 978-1-4939-6442-0
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