Download Supplementary Methods Sequencing of Multiplex PCR Amplicons

Supplementary Methods
Sequencing of Multiplex PCR Amplicons
Deep sequencing was performed using the Ion Torrent platform (Life Technologies),
according to the manufacturer’s specifications. Briefly, purified genomic DNA from the
FFPE sections were used for library construction with the Ion AmpliSeq Cancer Panel v2
(Life Technologies) that targets thousands of mutational hotspot regions of the 50 cancerassociated genes. In addition to the tumor DNA from PDAs, pool DNA samples were also
isolated using laser-captured micro-dissected PanIN lesions from 5 sections covering
normal pancreas. Fifteen to 50 ng DNA was subjected to generate libraries from each slide.
Emulsion PCR was performed with the Ion OneTouch system (Life Technologies) and the
amplicons were ligated to sequencing adaptors containing a unique bar code and specific
for the Ion Torrent sequencing platforms (Ion Xpress Barcode Adapters Kit; Life
Technologies). The quality of the obtained library was evaluated by the Agilent 2100
Bioanalyzer on-chip electrophoresis (Agilent Technologies). Sequencing was run on the Ion
Torrent Personal Genome Machine (PGM, Life Technologies) loaded with 318 chip as per
manufacturer’s protocol. Data analysis, including alignment to the hg19 human reference
genome and variant calling, was done using the Torrent Suite Software v.3 (Life
Technologies). Filtered variants were annotated using both the Ion Reporter software v1.2
(Life Techniologies) and the SnpEff software v.3.0 (alignments visually verified with the
Integrative Genomics Viewer; IGV v.2.1, Broad Institute).
Droplet digital PCR
To validate the mutations detected by deep sequencing, droplet digital PCR (ddPCR) for
KRAS and GNAS was conducted with the Bio-Rad QX200 system using custom
primer/probe set (see Supplementary Table 2). Genomic DNA (1-5 ng) was analyzed by
ddPCR using specific probes (PrimeTime Probes, IDT) for wild-type KRAS and GNAS as well
as KRAS p.G12D and GNAS p.R201C to detect and quantitate the mutations. PCR reactions
were run on Veriti thermal cycler (Applied Biosystems) incubating the plates at 95 °C for
10 minutes followed by 40 cycles of 94 °C for 30 seconds, 60 °C for 60 seconds, followed by
10-minute incubation at 98 °C. Plates were read on a QX200 droplet reader (Bio-Rad) using
QuantaSoft v1.6.6 software (Bio-Rad) to assess the number of droplets positive for wildtype and mutant DNA. The result was expressed as a fractional abundance of mutant to
total KRAS or GNAS molecules for each sample.