Ion Torrent PGM sequencing
Admixed melanoma and Burkitt's lymphoma (1:20) manual dissection
Library preparation was carried out using the Ion AmpliSeq Cancer Hotspot, Panel V2 and the Ion AmpliSeq Library Kit 2.0, according to the directions provided in the corresponding Ion Torrent User Guide. The Ampliseq Panel includes 207 primer sets covering ~2,800 COSMIC hotspot mutations in 50 cancer-related genes.  Briefly, individual DNA samples were subjected to multiplex PCR in a 96-well plate and library cleanup, followed by ligation to Ion Xpress Barcode Adapters 1-16.  Each barcoded library concentration was determined by qPCR with Ion Library TaqMan Quantitation Kit (Life Technologies) on a ViiA 7 Real Time PCR System.Each sequencing template, consisting of 4 barcoded libraries with equal concentration (5 ��__L at 20 pM concentration each), was amplified by emulsion PCR using the Ion PGM Template OT2 200 Kit on a OneTouch 2 instrument, following the instructions in the Ion Torrent User Guide. Following Emulsion PCR, the templated Ion Sphere particles (ISPs) were recovered and 1.0 ��__L aliquots of both enriched and unenriched ISPs were stained with SYBR Green Nucleic Acid Gel Stain (Life Technologies) and assessed for ISP quality and quantity on a Guava easyCyte 5 Flow Cytometer (Millipore). Approximately 20 million enriched ISPs were loaded into a 316 v2 chip and sequencing was performed on the PGM with the Ion PGM Sequencing 200 Kit v2.
NIH
ena-EXPERIMENT-NIH-10-02-2016-02:35:50:763-6
National Institutes of Health (NIH), Rockville, USA
ena-EXPERIMENT-NIH-10-02-2016-02:35:50:763-6