Duoprime

Cell-free DNA (cfDNA) was extracted with the MagMAX Cell-Free Total Nucleic Acid Isolation Kit on the KingFisher DuoPrime instrument according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 1.5 mL to 6 mL of plasma was incubated with Proteinase K and the Lysis/Binding solution, followed by the automated binding/washing, and elution steps. Elution was performed in 22 µL of the provided elution buffer, and the extracted cfDNA was stored at −20 °C.

Samples were deparaffinised in xylene (5 min), washed in 100% ethanol (2×5 min) and air-dried for 15 min. Tissue lysis was performed as per manufacturer instructions (Ion Torrent Genexus FFPE Combo kit) with Proteinase K in heat-blocks at 55ºC and 90ºC for 1 hour each prior to nucleic acid extraction. This off instrument pre-processing of samples took approximately 3 hours.
Following routine FFPE lysis protocol to obtain appropriate starting material, two methods of extraction were verified. The first was a semiautomated extraction with the KingFisher DuoPrime instrument with MagMAX FFPE DNA/RNA Ultra kit (Thermo Fisher, Scientific); the DNA/RNA concentration (n=181) was determined manually by fluorometric quantitation using the Qubit 2.0 Fluorimeter with Qubit DNA dsDNA BR Assay and RNA BR Assay kits (Reagecon).
The second extraction method streamlined the purification workflow with full automation using the Genexus Purification Instrument (GPI) on DNA and RNA over four runs (n=95). The hands-on time (10 min) was minimal, and the GPI extracted both DNA and RNA sequentially (Ion Torrent Genexus FFPE DNA/RNA Purification Combo Kit). The GPI has a built in Qubit for a fully automated workflow.
Quantified nucleic acids, with a minimum of 10 ng nucleic acid input, were prepared in an OPA output plate and manually transferred directly onto the Genexus Integrated sequencer.

Home stool sample collection kits were distributed to each participant. These kits included one sample tube containing RNAlater (5 mL; Thermofisher Scientific, Waltham, MA) as a sample preservative. Instructions for proper sample collection and storage were provided verbally and in print. Samples were submitted by mail or collected by a community research facilitator. Upon receipt, samples were stored at −20°C before nucleic acid purification.
Stool samples were processed via MagMAX Microbiome Ultra Nucleic Acid Isolation Kits (Thermo Fisher Scientific, Inc., Waltham, MA, United States) for simultaneous DNA and RNA extraction, using KingFisher Duo Prime automated extraction system. From each sample, 1–2 μL of purified DNA and RNA was analyzed using the Thermo Fisher NanoDrop Microvolume Spectrophotometer to assess sample quality and the Qubit Fluorometer for quantity (Thermo Fisher Scientific, Inc., Waltham, MA, United States). An overview of workflow methodology for sample processing and subsequent analyses are illustrated in Figure 1.