High-Throughput Amplicon Sequencing (ITS-LSU) of Hypocrealean Entomopathogenic Fungal Strains.

Data Acquisition

Context

This dataset was obtained as part of a research project integrating high-throughput sequencing (HTS) technology into fungal identification and barcoding. The specific experiment aimed to assess whether long-read HTS (PacBio SEQUEL I) could reliably identify entomopathogenic fungi (Hypocreales, Ascomycota) from our collections. Additionally, it enabled the characterization of intra-genomic variation in two ribosomal barcoding markers—the internal transcribed spacers (ITS) and the large subunit ribosomal (LSU) DNA—both widely used in fungal taxonomy and barcoding.

Protocol

Fungal materials and dna amplification

A total of 96 strains of entomopathogenic fungi (file: 1_list_sample_and_barcodes.xls) were obtained from culture collections and cultivated on potato dextrose agar (PDA) medium for one to four weeks. Mycelia were harvested, and DNA was extracted using a modified CTAB protocol. The entire ITS region (ITS1-5.8S-ITS2) and partial LSU DNA (D1-D2 domains) were co-amplified using universal primers ITS5 (forward) and LS5 (reverse). PCR amplifications were performed using two different DNA polymerases—DreamTaq DNA polymerase (Thermo Fisher) and Platinum SuperFi DNA polymerase (Invitrogen). Each primer was tagged with a barcode sequence, generating distinct combinations corresponding to different samples and polymerase conditions. PCR products from both polymerases were purified using the AMPure XP DNA purification kit. The DNA concentration of the purified products was quantified using Qubit™. All purified PCR products were then adjusted to a uniform concentration of approximately 15 ng/µl.

Amplicon High-Throughput Sequencing and Data Analysis

The amplicons were pooled as a basis to construct a unique sequencing library using the SMRTbell Template Prep Kit 1.0. The DNA library were validated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), quantifiedby Qubit 2.0 Fluorometer, and loaded on a PacBio SEQUEL I machine according to manufacturer’s instructions (Pacific Biosciences of California, Inc., California, USA). After obtaining the raw data (file: F.subreads.bam), Circular Consensus Sequences (CCS) were generated from subread sequences using the CCS tool, requiring a minimum of five subreads per sequence. Sample sequences were demultiplexed based on barcode information. To delineate the ITS and D1/D2 LSU regions, all sequences were bioinformatically cleaved at the ITS4 priming sites.