title: Rhizosphere metagenome from Humulus lupulus L.
design: The samples were processed and analyzed with the ZymoBIOMICS Targeted Sequencing Service (Zymo Research, Irvine, CA). DNA Extraction: One of two different DNA extraction kits was used depending on the sample type and sample volume. In most cases, the ZymoBIOMICS-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used to extract DNA using an automated platform. In some cases, ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA) was used for low biomass samples such as skin swabs, as it permits for a lower elution volume, resulting in more concentrated DNA samples. Targeted Library Preparation: Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S NGS Library Prep Kit (Zymo Research, Irvine, CA). In most cases, the bacterial 16S primers amplified the V3-V4 region of the 16S rRNA gene. These primers have been custom-designed by Zymo Research to provide the best coverage of the 16S gene while maintaining high sensitivity. Fungal ITS gene targeted sequencing was performed using the Quick-16S NGS Library Prep Kit with custom ITS2 primers substituted for 16S primers. The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with 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, Irvine, CA), then quantified with TapeStation(Agilent Technologies, Santa Clara, CA) and Qubit (Thermo Fisher Scientific, Waltham, WA). Control Samples: The ZymoBIOMICS Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction, if performed. The ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process. Sequencing: The final library was sequenced on Illumina Nextseq with a P1 reagent kit (600 cycles). The sequencing was performed with 30% PhiX spike-in.
library_name: zr13074_7V3V4