title: ITS rRNA amplicon high-throughput sequencing of fungi
design: After purification of PCR production, sequencing libraries were generated using NEB Next Ultra II DNA Library Prep Kit (New England Biolabs, Inc., USA) following the manufacturer's recommendations. The library quality was assessed by Nanodrop 2000 (ThermoFisher Scientific, Inc., USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA), and ABI StepOnePlus Real Time PCR System (Applied Biosystems, Inc., USA), successively. Deep sequencing was performed on Illumina Miseq/Nextseq 2000/Novaseq 6000 (Illumina, Inc., USA) platform at Beijing Allwegene Technology Co., Ltd.. After the run, image analysis, base calling and error estimation were performed using Illumina Analysis Pipeline Version 2.6 (Illumina, Inc., USA). Use Pear (v0.9.6) software to filter and splice raw data, and cut out the parts with low quality score ( 20) in the sequences. During splicing, the minimum overlap setting was 10bp, and the p-value setting was 0.0001. After splicing, Vsearch (v2.7.1) software was used to remove sequences with length less than 120 bp (ITS2 was 230 bp) and removed the chimeric sequence by uchime method according to the Unite Database. Qualified sequences were clustered into operational taxonomic units at a similarity threshold of 97% using Uparse algorithm of Vsearch (v2.7.6) sofware.
library_name: fungiIS