title: 16_Soil_16313R17
design: For 16S rRNA sequencing, ZymoBIOMICS-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used for DNA extraction. Positive controls included ZymoBIOMICS Microbial Community Standard (Zymo Research, Irvine, CA). For 16S rRNA sequencing, DNA libraries were prepared for using the Quick-16S Plus NGS Library Prep Kit (Zymo Research, Irvine, CA). ZymoBIOMIC 16S rRNA gene primers (forward CCTACGGGGNGGCWGCAG, reverse GACTACHVGGGTATCTAATCC) for V3V4 regions amplification of 16S rRNA genes. The sequencing library was prepared by PCR using real-time PCR thermocycler (QuantStudio 12K Flex, Applied Biosystems) to control cycles and limit PCR chimera formation. Quantification of final PCR products was performed using qPCR fluorescence readings before being pooled together at equal molarity. Pooled libraries were cleaned with DNA Clean & Concentrator kit (Zymo Research, Irvine, CA), and quantified with TapeStation (Agilent Technologies, Santa Clara, CA) and Invitrogen Qubit 1X dsDNA High-Sensitivity Assay Kits (Thermo Fisher Scientific, Waltham, WA). The final library was sequenced on Illumina NextSeq 2000 with a p1 (cat 20075294) reagent kit with 600 cycles. 16S amplicon sequencing was performed with 30% PhiX spike-in.