16S rRNA Seq of gut microbiome of fasted patients
A two-step PCR was chosen for library generation. For the first reaction, the 16S V3V4 region was amplified in 25 l quadruplicates. The Q5 Hot Start High-Fidelity DNA polymerase (New England BioLabs) was used with a primer pair first tested and characterized by Klindworth et al. (1) including overhanging Illumina adapter sequences (more detailed information and protocol steps can be found here:https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf.) The quadruplicate reactions were then pooled and DNA was cleaned up using a 1:1 ratio of solid-phase reversible immobilization (SPRI) on carboxylated paramagnetic beads (GE Healthcare, 65152105050250). For the second PCR reaction, Nextera XT barcodes (Illumina) were added. Again, libraries were cleaned up again using SPRI beads. Amount of nucleic acid was quantified with PicoGreen (P7589, Invitrogen, ThermoFisher Scientific). Equimolar amounts of respective samples were pooled and sequenced on the Illumina MiSeq using 2x300 bp chemistry (MiSeq Reagent Kit v3 MS-102-3003, Illumina).