Amplicon sequencing ITS region of stem endophytic fungi
By Illumina Miseq sequencing platform the fungal internal transcribed spacer (ITS1) region were sequenced. The primer sequences for PCR amplification of ITS1 were 5'- CTTGGTCATTTAGAGGAAGTAA-3'; 5'- GCTGCGTTCTTCATCGATGC-3', and the sequencing was performed with 300bp paired-end mode. The raw sequenced reads were trimmed with Trimmomatic v0.35. The quality trimmed paired-end reads were then overlapped and merged by FLASH v1.2.11. The merged sequences were further quality-filtering by QIIME v1.9.1 to remove sequences with length smaller than 200bp, maximum 2 nucleotide mismatch in primer matching, and sequences containing ambiguous bases or homologous bases. Subsequently, UCHIME v4.2 was used to identify and remove chimeric sequences.