Bioanalyser

RNA was extracted from 2.5 ml of blood using the PAXgene kit (Qiagen) and Reverse Transcriptase (RT) reactions were performed using the QuantiTect RT kit (Qiagen). Relative allelic expression of PIGY was measured by analysing cDNA using both Sanger sequencing and a 314 chip run on the Ion Torrent PGM platform. As the c.-540G>A variant is only 4 bp from the transcription start site, expression levels had to be measured indirectly using the T allele at rs3177413 (c.-222C>T) which familial transmission showed was in cis with c.-540G>A allele. RT-PCR primer sequences are shown in Table 1. The first round of amplification (25–30 cycles) and second round of amplification (15 cycles, with barcoding primers) were both performed with the FastStart Taq DNA polymerase kit (Roche). Samples were pooled, cleaned using Ampure beads, quantified using the BioAnalyser (Agilent) and diluted to 10 pM. Emulsion PCR and sequencing were carried out according to manufacturer instructions.
Quantitative PCR was performed using IQ SYBR Green Supermix (BIO-RAD) and the iQ5 Real-Time PCR Detection System (BIO-RAD) and the following primers: PIGY-V5-F 5′-AGGGATGTTCATCTCCAACCA-3′, PIGY-V5-R 5′-TGCGCATATCAGGCTTAGGA-3′, RPL30-F 5′-CAGACAAGGCAAAGCGAAAT-3′ and RPL30-R 5′-TGGACACCAGTTTTAGCCAAC-3′. PCRs were performed in triplicate, and the experiment was carried out three times.

Adapter-ligated indexed libraries were generated using the Illumina Nextera Rapid Capture kit (Illumina) from 50 ng of DNA as per manufacturer’s instructions. The libraries were quantified using a Qubit High Sensitivity dsDNA assay (Life Technologies). Five hundred nanograms of adapter-ligated barcoded DNA from each sample from each library were pooled into a capture pool of 12. Each capture pool was hybridised twice with enrichment probes for the exome. The fragment sizes of enriched libraries were assessed using a bioanalyser (Agilent Technologies, Folsom, CA, USA) and quantified using KAPA Library Quantification Kits (Kapa Biosystems, Wilmington, MA, USA).
Paired-end 125-bp sequencing runs were performed on an Illumina HiSeq 2500 instrument, aiming for a mean read depth coverage of 100 for the NA12878 dilution series.

This project was submitted to the Ethics Committees of the clinical centers taking part in the study and was approved by the National Institute of Cancer (INCa), following the recommendations of the French National Authority for Health (FNAH). Patient samples were processed according to the French Public Health Code (law n°2004–800, articles L. 1243–4 and R. 1243–61) and the biological resources center has been authorized (authorization number: AC-2008–700; Val d’Aurelle, ICM, Montpellier) to deliver human samples for scientific research. All patients were informed before surgery that their surgical specimens might be used for research purposes. They could refuse by completing an appropriate form; their tumor biopsies would then be destroyed. A total of 44 primary breast cancer samples were obtained from the Pathology Department. This study was reviewed and approved by the Montpellier Cancer Center - Val d’Aurelle Institutional Review Board and informed consent was obtained from all patients. Samples were systematically anonymized. RNAs were isolated from frozen tissues using the RNeasy Mini Kit (Qiagen S.A. France, Courtaboeuf, France) and their quality/quantity assessed on a Bioanalyser (Agilent, Santa Clara, CA, USA).