Technical replicate number 2 for sample SUBJECT_09-M12
16S rRNA gene sequencing targeting V3-V4 regions using a two-step PCR approach.
DNA was extracted from 200-300 mg of fecal material using mechanical lysis with bead-beating (FastPrep-24, 4x45 sec at speed 5) and the DNeasy Blood & Tissue Kit (Qiagen). DNA concentrations were measured by PicoGreen assay. Library preparation was performed using a two-step PCR targeting the V3-V4 hypervariable regions of the 16S rRNA gene with primers 341F and 805R. The first PCR included 20 cycles, followed by a second PCR of 10 cycles for indexing.