TABLE I.

The standard operating procedures defined and observed for the study

Assay	Procedure and description
Specimen collection, preparation, and quality assessment
Specimen collection and new case entry Describes how and when to collect new cases from the clinical lab (histopathology), how to record information, how to process cases, and where to store them. Purification of genomic DNA and total RNA from formalin-fixed paraffin-embedded tissue sections Describes how to extract DNA and RNA from tissue sections that have been formalin-fixed and paraffin-embedded. Quantifying, labelling, and storing RNA Describes how to quantify RNA, how to correctly design and print labels, where to store the original extraction case, and how to enter RNA extraction information into the tracking database. Quantifying, labelling, and storing DNA Describes how to quantify DNA, how to correctly design and print labels, where to store the original extraction case, and how to enter DNA extraction information into the tracking database. Preparing tumour DNA dilutions for targeted capture sequencing Describes how to prepare, label, and store 50 ng/μL dilutions for targeted capture sequencing. DNA quality assessment Describes how to assess and estimate the degree of degradation in DNA extracted from formalin-fixed, paraffin-embedded tissue. RNA quality assessment Describes how to assess the quality of RNA. Identifying and tracking peripheral blood DNA cases Describes how to identify, locate, and track case peripheral blood DNA cases in the tracking database. Preparing peripheral blood DNA dilutions for targeted capture sequencing Describes how to prepare, label and store dilutions for targeted capture sequencing.
Digital gene expression profiling
10. NanoString Technologies nCounter Gene Expression Assaya hybridization procedure Describes how to perform the NanoString Technologies nCounter Gene Expression Assay hybridization using total RNA extracted from formalin-fixed, paraffin-embedded tissue sections. 11. NanoString Technologies nCounter prep station operation Describes how to operate the NanoString Technologies nCounter prep station for purification and immobilization of post-hybridized nCounter assay cases to an nCounter cartridge allowing for subsequent data collection on the nCounter Digital Analyzer. 12. NanoString Technologies nCounter Digital Analyzera operation Describes how to operate the NanoString Technologies nCounter Digital Analyzer for data collection.
Fluorescence in situ hybridization (FISH)
13. FISH protocol for paraffin-embedded tissue Describes how to perform FISH on paraffin-embedded tissues. 14. FISH analysis Describes how to automatically score a tissue section using the Metaferb semi-automatic scoring software.
Targeted capture sequencing and bioinformatics analysis
15. Illumina Multiplex Sequencingc Describes an optimized protocol for Illumina paired-end multiplex library preparation using the SureSelect XT2 Library Prep and Capture Systemc. 16. Sequence analysis for mutation calling Describes the processing and analysis of sequence data to identify high-confidence somatic variants (“mutation calls”).

^aNanoString Technologies, Seattle, WA, U.S.A.

^bMetaSystems, Heidelberg, Germany.

^cIllumina, San Diego, CA, U.S.A.