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Anchored multiplex PCR for targeted next-generation sequencing


We describe a rapid target enrichment method for next-generation sequencing, termed anchored multiplex PCR (AMP), that is compatible with low nucleic acid input from formalin-fixed paraffin-embedded (FFPE) specimens. AMP is effective in detecting gene rearrangements (without prior knowledge of the fusion partners), single nucleotide variants, insertions, deletions and copy number changes. Validation of a gene rearrangement panel using 319 FFPE samples showed 100% sensitivity (95% confidence limit: 96.5–100%) and 100% specificity (95% confidence limit: 99.3–100%) compared with reference assays. On the basis of our experience with performing AMP on 986 clinical FFPE samples, we show its potential as both a robust clinical assay and a powerful discovery tool, which we used to identify new therapeutically important gene fusions: ARHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma, MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1 and RUFY2-RET in lung cancer, FGFR2-CREB5 in cholangiocarcinoma and PPL-NTRK1 in thyroid carcinoma. AMP is a scalable and efficient next-generation sequencing target enrichment method for research and clinical applications.

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Figure 1: AMP for targeted RNA and DNA sequencing.
Figure 2: Targeted RNA sequencing for rearrangement using AMP for the detection of unknown 5′ or 3′ gene fusion partners.
Figure 3: Targeted DNA sequencing using AMP for the detection of single nucleotide variants, insertions, deletions and copy number variants.


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This study was supported by a US National Institutes of Health grant (R21CA161590) to A.J.I. and a postdoctoral fellowship from the Swedish Research Council (350-2012-368) to Z.Z.

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Authors and Affiliations



Z.Z., M.L., B.Z., Y.C., D.P., K.D.L., J.C., H.E.R., H.S.S., J.C., W.P. & J.A.E. conducted the experiments; Z.Z. and L.P.L. conducted the data analyses; Z.Z. and L.P.L. wrote the manuscript; A.J.I. supervised the project.

Corresponding authors

Correspondence to A John Iafrate or Long Phi Le.

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Competing interests

A provisional patent has been filed by A.J.I., L.P.L. and Z.Z. for the anchored multiplex PCR technology with the US Patent Office. A.J.I., L.P.L. and Z.Z. are equity holders in Enzymatics, Inc., a licensee of the technology.

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Zheng, Z., Liebers, M., Zhelyazkova, B. et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med 20, 1479–1484 (2014).

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