title: WCX-18s
design: 18S Library Preparation and Illumina MiSeq Sequencing  Next generation sequencing library preparations and Illumina MiSeq sequencing were   conducted at GENEWIZ, Inc. (Beijing, China).  DNA samples were quantified using a Qubit 2.0   Fluorometer (Invitrogen, Carlsbad, CA) and DNA quality was checked on a 0.8% agarose gel.   5-50 ng DNA was used to generate amplicons using a panel of primers designed by GENEWIZ  (GENEWIZ, Inc., South Plainfield, NJ, USA).  18S sequencing  Multiple oligonucleotide primers were designed to anneal to the relatively conserved sequences   spanning fungi 18s rDNA hypervariable regions v4 and v5  to amplify fragments less than 500   bp. The 3' part of v4 and full v5 regions were amplified using forward primer containing   sequence "TTAGCATGGAATAATRRAAT" and reverse primer containing sequence   "TCTGGACCTGGTGAGT". Besides the 18S target-specific sequences, the primers also   contain adaptor sequences allowing uniform amplification of the library with high complexity   ready for downstream NGS sequencing on Illumina Miseq platform.  DNA libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto,   CA, USA), and quantified by Qubit and real time PCR (Applied Biosystems, Carlsbad, CA, USA).    DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to   manufacturer’s instructions (Illumina, San Diego, CA, USA).  Sequencing was performed using   a  2x300 paired-end (PE) configuration; image analysis and base calling were conducted by the   MiSeq Control Software (MCS) on the MiSeq instrument.