Taq kapa hifi hotstart readymix

From the ileal and cecal digesta 200 mg of samples were lysated with MagNA Lyser (Roche, Burges Hill, UK) previously to DNA extraction. DNA extraction was performed with PowerMicrobiome™ RNA isolation kit (MO BIO, Carlsbad, CA, USA) following the manufacturer's instructions with some modi cations, such as omitting the β-mercaptoethanol and DNase I steps. The concentration of the extracted prokaryotic DNA in each sample was calculated by qPCR with 926F (De Gregoris et al., 2011) and 1027R (Claesson et al., 2009) primers at a concentration of 0.4µM in iQ SYBRgreen Supermix qPCR (Bio-Rad Laboratories Inc., Hercules, CA, USA). PCR was carried out with Universal primers 341F-785R of V3-V4 regions to amplify 16S rRNA in a dual-index sequencing strategy according to Kozich et al. (2013) with Taq KAPA HiFi Hotstart ReadyMix (Kapa Biosystems, Woburn, MA, USA) and 12.5 ng bacterial DNA to reduce PCR bias. Four PCR products from the same sample were pooled before cleaning up step with QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Negative controls and MOCK communities were PCR and included in the sequencing. The library was sequenced on an Illumina HiSeq platform 2×300 paired end. All reagents used were molecular grade.