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.
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
Zongli
0000000348494903
Z
Zheng
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Matthew
M
Liebers
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Boryana
B
Zhelyazkova
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Yi
Y
Cao
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Divya
D
Panditi
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Kerry D
KD
Lynch
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Neurosurgery, Shanghai Changzheng Hospital, Shanghai, China.
Juxiang
J
Chen
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Hayley E
HE
Robinson
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.
Hyo Sup
HS
Shim
Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Juliann
J
Chmielecki
Vanderbilt University Medical Center, Nashville, Tennessee, USA.
William
W
Pao
Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA.
Jeffrey A
JA
Engelman
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA.
A John
AJ
Iafrate
1] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA.
Long Phi
LP
Le
D001650:Bile Duct Neoplasms / Q000235:genetics*; D018281:Cholangiocarcinoma / Q000235:genetics*; D050939:Gene Fusion / Q000235:genetics*; D015321:Gene Rearrangement / Q000235:genetics*; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013964:Thyroid Neoplasms / Q000235:genetics*