Sample collection

Buccal sample

To ensure adequate DNA yield for molecular profiling of buccal cell samples, we utilised four Qiagen OmniSwab devices (#WB100035), with two devices used per side. The cheeks were swabbed with external counterpressure and swab heads were placed in a single tube and stored at -20C until DNA extraction for subsequent epigenetic analysis.

Blood sample

Blood was drawn by trained personnel and collected into various devices depending on intended use (see publication)

Cervical sample

Samples were collected during the study visit using a speculum. The brush was rotated five times and placed in the Hologic ThinPrep® collection device. The device was transported to the lab and samples were pelleted. The pellet was washed once using phosphate buffered saline, and subsequently stored at -80C until DNA extraction for subsequent epigenetic analysis.

Saliva sample

Participants were asked to collect two saliva samples at home on the day of the study visit: native and DNA/RNA stabilised. Participants were asked to not eat, drink, smoke, or use oral hygiene products for one hour prior to sample collection, and ideally collect samples prior to brushing teeth. For the native sample, the mouth was rinsed with water and 5 minutes later, 2 mL of saliva were placed in a collection tube. The sample was placed in the provided cooling bag until the study visit, where it was aliquoted and stored at -80C until further processing. The stabilised sample was placed into a Zymo Research DNA/RNA Shield Saliva Collection Kit tube (#R1210-E) and inverted 10x. At the study visit, it was aliquoted and stored at -20C until further processing.

Urine sample

Participants were asked to collect a urine sample at home on the day of the study visit. Participants collected morning urine and placed the collected sample in the cooling bag until the study visit, where it was centrifuged to separate the supernatant and pellet. The supernatant was aliquoted and stored at -80C until further processing. The pellet was stored at -80C until further processing.

Fecal sample

Participants were asked to collect two fecal samples at home as close as possible to the day of the study visit: native and DNA/RNA stabilised. The native sample was placed in the appropriate tube and placed in the provided cooling bag until the study visit, where it was aliquoted and stored at -80C until further processing. The stabilised sample was placed in a DNA stabilisation solution (Invitek Stool Collection Tubes with DNA Stabilizer, #1038111), aliquoted at the study visit, and immediately stored at -80C until further processing.

Skin biopsy

Participants were invited to provide optional skin biopsies at baseline and 6 months. Participants provided a separate informed consent for skin biopsies. Skin barrier measurements using tape strips and non-invasive measurements of transepidermal water loss (TEWL) were conducted, and subsequently two to three 6 or 8 mm biopsies were taken from the skin of the inner arm or leg. Before biopsy, the area was be disinfected and anaesthetised. Once the local anaesthetic has taken affect a 6 or 8 mm diameter deep punch biopsy was be performed and the tissue transferred to a sample vial. The biopsy site was be closed with sutures which were removed 10-14 days later. Instructions for volunteers on skin care were be provided. Samples were placed in liquid nitrogen or formaldehyde for immunohistochemistry analysis.