Gs junior system

Isolated DNA (350–976 ng) from all FFPE specimens was subjected to analyses by NGS using a Roche GS Junior System to detect BRAFV600 mutations on exon 15. The NGS procedure was done according to the manufacturer's specifications [16 (link)]. Amplicon processing, library preparation and emulsion PCR were done according to the manufacturer's directions for the GS Junior Titanium Series (Roche). Around 500,000 enriched beads were loaded on a 454 Junior Sequencer (Roche, Basel, Switzerland). Demultiplexing and variant calling was done with the Amplicon Variant Analyzer v2.7 software from Roche. The average sequencing coverage of BRAF was >5000. The presence of a BRAFV600 mutation was defined as the presence of a non-reference base in a minimum of 3% of reads.

TGFBR1 and TGFBR2 primers were designed and validated by Fluidigm (Fluidigm Corporation, San Francisco, CA) as per recommended guidelines for Roche Titanium sequencing (Roche, Mannheim, Germany). Primers for NOTCH1, NOTCH2, TP53, CDKN2A, HRAS, KRAS and NRAS were previously described6 (link) and all primer sequences are listed in Supplementary Data 27. Each primer included sample-specific Fluidigm 454 barcode primer and adapter sequences. Sequencing was performed in the same manner as our previous study56 (link). Briefly, for thermal cycling a Fluidigm FC1 Cycler was used. The libraries were normalized and pooled before purification using Agencourt AMPure XP system (Beckman, UK). Library components were clonally amplified utilising the GSJunior emPCR Lib-A Kit (Roche) by inputting one molecule of library DNA per capture bead. Pyrosequencing was done using the GS Junior system (Roche/454 Life Sciences).

Each PCR primer consisted of a specific adaptor (5′-CGTATCGCCTCCCTCGCGCCATCAG-3′ for forward primers and 5′CTATGCGCCTTGCCAGCCCGCTCAG-3′ for reverse primers), a sample-specific multiplex identifier (MID) sequence, and a specific amplification sequence for PCR (Table S2).
We obtained three amplicons for each sample. Each amplicon (22.5 μl) was diluted with an equal volume of water and purified with AMPure beads (72 μl), before quantification with a Qubit^®; ds DNA HS Assay kit (Life Technologies) on a Qubit 3.0 instrument. For each gene, the PCR amplicons from 16 samples were pooled in equimolar concentrations and used to prepare an equimolar mixture of 10⁷ molecules. This pool was subjected to clonal amplification on capture beads by emulsion PCR. In total, 500,000 beads enriched in DNA were deposited into the wells of a PicoTiter plate device and subjected to pyrosequencing in both directions with the GS Junior system (Roche). After a 10-h run, total reads were analyzed with GS amplicon variant analyzer (AVA) software (Roche), filtered and assigned, by demultiplexing, to the correct patient sample on the basis of MID correspondence.