title: Microbiota profile of Melanaphis sacchari
design: The 16S hypervariable region of ribosomal subunit was amplified from 5 ng of microbial DNA using the Ion 16S Metagenomics kit (ThermoFisher Scientific) on two polymerase chain reactions (PCR) with the primer sets V1, V2, V4, V8 and V3, and V6-V7 and V9. The amplicons were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc, Atlanta, GA, USA) and later quantified using Agilent High Sensitivity DNA (Agilent Technologies, Santa Clara, CA, USA) in an Agilent 2100 Bioanalyzer. The DNA libraries were developed using fifty nanograms of combined amplicons with the Ion Plus Fragment Library Kit (ThermoFisher Scientific) according to the manufacturers specifications. The DNA libraries were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) and then quantified with the Agilent High Sensitivity DNA kit (Agilent Technologies) in an Agilent 2100 Bioanalyzer. Each sample was adjusted to a concentration of 26 pmol, subjected to PCR in an emulsion with the Ion PGM Hi-Q OT2 kit (ThermoFisher Scientific) in a OneTouch2 system (Life Technologies, Grand Island, NY, USA), and subsequently enriched using the Dynabeads MyOne Streptavidin C1 Beads kit in an Ion OneTouchES (Life Technologies). The sequencing of each library was performed with an Ion PGM System using the Ion PGM Hi-Q Sequencing kit (Thermo Fisher Scientific) in a 314 V2 chip. Base-calling was performed with Torrent Suite version 4.4.2 (Life Technologies) and FastQC version 3.4.1.1 (Life Technologies) to generate FASTQ files for each sample. Reference FASTA=MicroSEQ 16S Reference Library v2013.1 and Greengenes v13.5 (Life Technologies) curated databases.