Zymobiomics targetedsequencing service for microbiome analysis

The gut microbiome of GF-transplanted male mice (n = 17) was analyzed by ZymoBIOMICS® Targeted Sequencing Service for Microbiome Analysis (Zymo Research, Irvine, CA). The ZymoBIOMICS®-96 MagBead DNA Kit was used to extract DNA, using an automated platform. Bacterial 16S ribosomal RNA gene-targeted sequencing was performed by using the Quick-16S™ NGS Library Prep Kit (Zymo Research). The bacterial 16S primers amplified the V3–V4 region of the 16S rRNA gene. The sequencing library was prepared by using an innovative process in which PCR reactions were performed in RT-PCR machines to control cycles and, therefore, limit PCR chimera formation. The final PCR products were quantified by qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research) and then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific). The final library was sequenced on Illumina® MiSeq™ with a v3 Reagent Kit (600 cycles). The sequencing was performed with a 10% PhiX spike-in.