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Nat Med. 2014 Dec;20(12):1479-84. doi: 10.1038/nm.3729. Epub 2014 Nov 10.

Anchored multiplex PCR for targeted next-generation sequencing.

Author information

1
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
2
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
3
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Neurosurgery, Shanghai Changzheng Hospital, Shanghai, China.
4
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.
5
Vanderbilt University Medical Center, Nashville, Tennessee, USA.
6
Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA.
7
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

Abstract

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.

PMID:
25384085
DOI:
10.1038/nm.3729
[Indexed for MEDLINE]
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