CRC serum
DNA was isolated from serum samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Then, metagenomic analysis of serum microbiota was performed via the amplification and sequencing of the bacterial 16S rRNA gene using the Ion Torrent sequencing technology and the reagents from Life Technologies S.A. (a part of Thermo Fisher Scientific), as described previously. Briefly, DNA was quantified using the Invitrogen Qubit 3 Fluorometer with the dsDNA HS (high sensitivity) Assay (Thermo Fisher Scientific, Waltham, MA, USA).  A known amount of DNA extracted from each sample (5 ng) underwent parallel polymerase chain reactions (PCRs). According to the Ion 16SMetagenomics Kit protocol, two distinct primer sets were used to amplify seven hypervariable regions of the gene (V2, V4, V8, and V3, V67, V9, respectively). Subsequently, the resulting amplicons were quantified, pooled equimolarly for each sample, and purified using Agencourt AMPure XP reagent beads (Beckman Coulter, Pasadena, CA, USA). Barcoded libraries were generated using the Ion Plus Fragment Library Kit and the Ion Xpress Barcode Adapters. After quantification using the Qubit 3 Fluorometer libraries were set to 22 pM, pooled, and subjected to emulsion PCR with the Ion OneTouch 2 System and the Ion 520 and 530 KitOT2. Template-positive ion sphere particles were collected, washed, enriched using the Ion OneTouchES Instrument OT2, and finally loaded onto an Ion 530 Chip for sequencing with the Ion S5 System.  Two extraction blanks were also included, which underwent all extraction and sequencing steps along with the samples and were subsequently used for the correction of sample lectures.