Oragene protocol

Genomic DNA was extracted from peripheral white blood cells using a modified protocol of the salting-out technique [25 (link)], and from saliva using an alcohol precipitation method following the Oragene protocol (DNA Genotek). The quality and quantity were assessed using the Nanodrop 2000c UV Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The 12 most genetically informative relatives from the pedigree were selected for WGS, which also included an unaffected healthy control to aid in gene variant filtering. WGS was carried out on the BGISEQ-500 sequencing platform at the Beijing Genomics Institute (BGI) Tech Solutions Co. Limited, Hong Kong, China using the DNA Nanoballs technology for the generation of the library and the combined primer anchor synthesis for sequencing of nucleotides.

We mailed an Oragene DNA self-collection kit (DNA Genotek, Kanata, ON, Canada) to the participant and return postage-paid mailing materials. Of the 371 women eligible for the current study, 328 women provided sufficient amount of samples for DNA extraction. We extracted DNA using the Oragene protocol (DNA Genotek).

The collection of specimens has been described previously [32 (link)]. Briefly, cases and controls provided a blood sample and completed a brief questionnaire at the time of blood draw, which updated breast and reproductive and gynecologic history and several lifestyle factors. Women who declined providing blood provided saliva in Oragene DNA self-collection kits (DNA Genotek, Kanata, ON, Canada). All biological specimens were sent overnight to the UCI laboratory. DNA was extracted from blood clots using Qiagen Clotspin Baskets and DNA QIAmp DNA Blood Maxi Kits (Qiagen, Inc., Valencia, CA, USA) in accordance with Qiagen protocols. DNA was extracted from saliva samples using the Oragene protocol (DNA Genotek).