Abridged adapter primers increase the target scope of Hi-Plex

T Nguyen-Dumont; F Hammet; M Mahmoodi; BJ Pope; GG Giles; JL Hopper; MC Southey; DJ Park

Journal article

Abridged adapter primers increase the target scope of Hi-Plex

T Nguyen-Dumont, F Hammet, M Mahmoodi, BJ Pope, GG Giles, JL Hopper, MC Southey, DJ Park

Biotechniques | BIOTECHNIQUES OFFICE | Published : 2015

DOI: 10.2144/000114247

Abstract

Previously, we reported Hi-Plex, an amplicon-based method for targeted massively parallel sequencing capable of generating 60 amplicons simultaneously. In further experiments, however, we found our approach did not scale to higher amplicon numbers. Here, we report a modification to the original Hi-Plex protocol that includes the use of abridged adapter oligonucleotides as universal primers (bridge primers) in the initial PCR mixture. Full-length adapter primers (indexing primers) are included only during latter stages of thermal cycling with concomitant application of elevated annealing temperatures. Using this approach, we demonstrate the application of Hi-Plex across a broad range of ampli..

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University of Melbourne Researchers

Tu Nguyen Author

Bernard Pope Author

Melissa Southey Author

Daniel Park Author

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Awarded by National Cancer Institute

Funding Acknowledgements

This work was supported by the Australian National Health and Medical Research Council (NHMRC) (APP1025879 and APP1029974), The Cancer Council Victoria (APP1066612), a Victorian Life Sciences Computation Initiative (VLSCI) grant (VR0182) on its Peak Computing Facility at The University of Melbourne, the National Institutes of Health (USA) (RO1CA155767) and the Victorian Breast Cancer Research Consortium (VBCRC). TN-D is a Susan G. Komen for the Cure Postdoctoral Fellow. MCS is a Senior Research Fellow of the NHMRC. We thank the Australian Breast Cancer Family Study (ABCFS, Principal Investigator John Hopper) for providing the cell-line derived DNA. This paper is subject to the NIH Public Access Policy.