ITS1-5.8s_ITS2 Fungal Sequences and Search Results .xlsx
The ITS1‑5.8s‑ITS2 region of the extracted rDNA of fungal isolates was chosen to be amplified based on its success in identifying a wide range of fungal species [53]. For DNA amplification, 10.0 mL of REDExtract-N-Amp™ PCR Ready Mix; 7.8 mL of PCR-grade H2O; 0.8 mL of 10 mM forward primer (ITS1, sequence TCCGTAGGTGAACCTGCGG); 0.8 mL of 10 mM reverse primer (ITS4, sequence TCCTCCGCTTATTGATATGC); and 1.0 mL of extracted fungal DNA sample were added to a 200 mL Eppendorf PCR tube. The same method was used to prepare the negative control. PCR amplification was performed with a preliminary step of polymerase activation at 94 oC for 2 minutes; 35 cycles of denaturation at 94 oC for 30 seconds, annealing at 51 oC for 20 seconds, and extension at 77 oC for 1 minute; and a final extension step at 72 oC for 8 minutes, using the Eppendorf Vapo. Protect ™ Mastercycler® Pro S.
To confirm a successful fungal DNA extraction and amplification, 4 mL of the amplified fungal rDNA product of the PCR reaction was loaded onto a 1 % (w/v) agarose gel in a 1x Tris/Borate/EDTA (TBE) buffer, and 1 mL cyanide dye SYBR® DNA gel stain was added for visualisation purposes. One kilobase (1kb) plus DNA ladder (5 mL) and 5 mL of the negative control were also loaded onto the agarose gel. Following the completion of gel electrophoresis, PCR products were visualised with the GelDocTM XR Plus System (BIO‑RAD, USA). The 1kb plus DNA ladder was used to determine the size of the amplified fungal DNA fragments using the Gelanalyzer 2010a quantification programme. The fungal rDNA fragments of the ITS1‑5.8s‑ITS2 region obtained from PCR were then transferred to the Centre of Genomics, Proteomics and Metabolomics DNA sequencing facility for sequencing.
Capillary Electrophoresis DNA Sequencing (Sanger Sequencing) was used to obtain the DNA sequences of the amplified ITS1‑5.8s‑ITS2 region. Each sample containing fungal DNA template had two reactions performed, one for each primer and were mixed with the ABI PRISMTM BIG DYE Terminator Sequencing Kit version 3.1 (ThermoFisher Scientific) containing DNA polymerase enzyme, a buffer, four DNA nucleotides and four chain-terminating dideoxy nucleotides with fluorescent dyes. The samples were then subjected to cycle sequencing on the thermal cycler Applied Biosystems GeneAmp® PCR System 9700 using standard cycling conditions: a preliminary step of polymerase activation at 96 oC for 1 minute; 25 cycles of denaturation at 96 oC for 10 seconds, annealing at 50 oC for 5 seconds, and extension at 60 oC for 4 minutes. Following the cycle sequencing, the samples were purified using Agencourt® CleanSEQ® magnetic beads in order to remove the excess fluorescent dyes, nucleotides, salts and other contaminants. The remaining purified DNA samples were then separated by size by capillary electrophoresis with the ABI PRISMTM 3130XL Genetic Analyzer using 50 cm capillaries and POP7 polymer. The final data output of the ITS‑5.8s‑ITS2 region DNA sequences was based on the detection of the attached fluorescent dyes excited by a laser.
Geneious programme version 11.1.5 (www.geneious.com) was used to analyse the raw data [54]. The data included both forward and reverse rDNA sequences for each fungal isolate. These sequences were aligned and ends showing poor quality reads were trimmed, to obtain a consensus sequence. A tool within the Geneious programme, BLAST (Basic Local Alignment Search Tool) developed by Altschul et al. [55], optimised for fast and high similarity search (MegaBLAST version), was used to compare the consensus query sequence with known DNA sequences in GenBank (NCBI genetic sequence database), EMBL (European Molecular Biology Laboratory), DDBJ (DNA DataBank of Japan) and PDB (Protein Data Bank, Worldwide). The search results included: grade percentage score showing combinatorial results of the query input sequence coverage, expectation-value (e-value) and identity value for each hit against the database; identities match and percentage score indicating the extent to which the query DNA sequence matched the database nucleotide sequence; and bit-score showing the quality of alignment and measuring sequence similarity [56]. The higher the score of each result, the higher the certainty of identification of the fungal species. Grade percentage score of >98 % was considered as correct genomic identification.
