Massarray system

For the targeted detection of somatic mutations in CTCs and tissue we used the iPLEX HS technology in conjunction with the MALDI-TOF based MassARRAY System (Agena Bioscience, San Diego, California). Here we used the Lung Panel covering 70 mutations in five key oncogenes (BRAF, EGFR, ERBB2, KRAS and PIK3CA) implicated in disease progression and therapy response. A mutation signal produced using iPLEX HS chemistry can be reliably detected by the MassARRAY System at about 1% VAF^{23 (link)}.

We performed DNA extraction and purification from peripheral blood samples according to the instructions of the kit (GoldMag Co. Ltd. Xi’an, China). The purified DNA was stored in the refrigerator. All primers in the present study were designed by MassARRAY Assay Design software. We used the MassARRAY system (Agena, San Diego, CA, U.S.A.) to genotype SNPs.

Buccal saliva samples were collected via mouth swabs with participants instructed to brush the edge of a soft tip swab along the insides of their cheek and gums for 30 s [58 (link),59 ]. Players were asked not to consume coffee, alcohol, or food two hours prior to saliva collection. Collected samples were labelled with a numeric code for de-identification and were sent to the Australian Genome Research Facility (AGRF; Brisbane, QLD, Australia; NATA 17025) for DNA extraction and genotyping using the Agena Bioscience MassARRAY system (AGRF). A summary of the genetic variants investigated in this study is presented in Table 1.

To assess the relation between systemic markers and tumour molecular alteration, we performed somatic mutation analysis of seven genes associated with colorectal cancer (KRAS, BRAF, NRAS, APC, PIK3CA, PTEN and TP53).
Mutation detection was performed using iPLEX^® Pro reagent on MassARRAY^® System (Agena Bioscience, CA). The 53-hotspot target mutation sites of the following genes: APC, BRAF, KRAS, NRAS, PIK3CA, PTEN and TP53 for colon cancer were designed (Supplementary Table 1). About 10 ng of genomic DNA was amplified using HotStarTaq polymerase (Qiagen, Valencia, CA), 100 nm of primers and 0.5 mM dNTPs (Invitrogen, Inc.) on 9700 thermal cycler (Thermoscientific, MA, USA). PCR products were treated with SAP (Shrimp Alkaline Phosphatase) enzyme. Single-base extension reaction was performed followed SAP treatment. The final product was cleaned with resin, and 16-nl product was transferred to a spectrochip using Nanodispenser RS 1000 (Agena Bioscience, CA). Finally, one nucleotide difference was detected using MALDI-TOF, and allele frequency was calculated using TYPER v4.0 software. This work was supported by the Genomics Core Facility in the National Cancer Center Korea.

Buccal saliva samples were collected via mouth swabs with participants instructed to brush the edge of a soft tip swab along the insides of their cheek and gums for 30 s [52 (link)–54 (link)]. Samples were collected before a pre-season training session and players were asked not to consume coffee, alcohol, or food for two hours prior to saliva collection. Collected samples were labelled with a numeric code for de-identification and were sent to the Australian Genome Research Facility (AGRF; Brisbane, QLD, Australia; NATA 17025) for DNA extraction and genotyping using Agena Bioscience MassARRAY system (AGRF; Brisbane, QLD, Australia). Genetic variants examined were within the following genes: ACTN3 (rs1815739), CCL2 (rs2857656), COL1A1 (rs1800012), COL5A1 (rs12722), COL12A1 (rs970547), EMILIN1 (rs2289360), IGF2 (rs3213221), NOGGIN (rs1372857), SMAD6 (rs2053423), with all genotypes being within Hardy–Weinberg equilibrium (HWE), as previously reported [52 (link)].