Prepit l2preagent

Genomic DNA from human saliva samples was collected in Oragene^® DNA collection kits and was then isolated using the prepIT.L2Preagent (cat # PT-L2P-5, DNA Genotek Inc., Canada) and precipitated with ethanol according to manufacturer’s instructions. The DNA samples were genotyped for BDNF (the single nucleotide polymorphism rs6265) using the TaqMan SNP Genotyping Assay (C__11592758_10) designed by Thermo Fisher Scientific. Primers and probes were mixed with TaqMan^® Universal PCR Master Mix (Thermo Fisher Scientific). 4.5 μl of genomic DNA (2.5 ng/μl) was transferred in triplicate to a 384-well plate, with each well containing 5.5 μl of the PCR mixture. The PCR reaction was performed following a protocol provided by ABI. The allele was discriminated by post-PCR plate reading on the ViiA™ 7 System. Data were processed using the ViiA™ 7 Software (Thermo Fisher Scientific).

Brain DNA was extracted using AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Hilden, Germany) after disrupting and homogenizing 10 mg RNAlater® preserved brain tissues by a rotor–stator (speed: 6.5 m/sec, running time: 45 s, FastPrep FP120J-100, Savant Instruments, Inc.) with tissue homogenization beads (Lysing Matrix D, 2 mL, MP biomedicals, LLC, CA, US). Although this preprocessing was originally for RNA extraction, we followed the instructions in the RiboPure^TM RNA Purification Kit (Thermo Fisher Scientific, Inc., MA, US) for preprocessing for DNA extraction using RNAlater® preserved blood samples. In brief, the RNAlater® preserved blood samples (720 μL) were centrifuged for 1 min at 16 000 ×g, and the supernatant was removed. Then, 200 μL of PBS was pipetted in and mixed, and centrifugation and supernatant removal were repeated. Finally, 200 μL of PBS was added and pipetted together before being used for DNA extraction. QIAamp DNA Mini kit (QIAGEN, Hilden, Germany) was used to extract DNA from blood and buccal, and the protocols for each tissue type were followed. Meanwhile, the prepIT®•L2P reagent (DNA Genotek Inc., Ottawa, CA) was used to extract DNA from saliva. The DNA yield was determined using the Qubit^TM dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Inc., MA, US) [8 (link)].

DNA isolation was performed according to the manufacturer’s instructions for buffy coat (FlexiGene DNA kit, Qiagen, or Promega’s Maxwell 16 Blood DNA Purification Kit) in SGBCC and MyBrCa, and G-DEX(TM) II Genomic DNA extraction kit (Intron) in KOHBRA. DNA isolation for saliva samples (only in SGBCC) was performed using Oragene and prepIT•L2P reagent, DNA Genotek.
Target-enriched sequencing libraries of germline DNA for the breast cancer cases and controls were prepared at the Centre for Cancer Genetic Epidemiology (University of Cambridge) as part of a larger effort (Breast Cancer Risk after Diagnostic Gene Sequencing, BRIDGES, https://bridges-research.eu) [13 (link)]. The gene panel, which was developed as part of the BRIDGES initiative, included coding sequences and intron/exon boundaries for a total of 34 genes for which there was prior evidence of association with breast cancer risk, including genes offered on commercial panels for breast cancer in early 2016 (Additional file 1: Table S1). The targeted sequencing workgroup in BRIDGES first performed rare variant detection in preliminary gene panels combined with data from whole-exome sequencing datasets before arriving at the 34 genes selected for the BRIDGES panel. Details of the library preparation, sequencing, variant calling, and quality control methods are given in Dorling et al. [13 (link)].