Characterization of the Castanopsis carlesii Deadwood Mycobiome by Pacbio Sequencing of the Full-Length Fungal Nuclear Ribosomal Internal Transcribed Spacer (ITS)

Abstract

Short-read next generation sequencing (NGS) platforms can easily and quickly generate thousands to hundreds of thousands of sequences per sample. However, the limited length of these sequences can cause problems during fungal taxonomic identification. Here we validate the use of Pacbio sequencing, a long-read NGS method, for characterizing the fungal community (mycobiome) of Castanopsis carlesii deadwood. We report the successful use of Pacbio sequencing to generate long-read sequences of the full-length (500–780 bp) fungal ITS regions of the C. carlesii mycobiome. Our results show that the studied deadwood mycobiome is taxonomically and functionally diverse, with an average of 85 fungal OTUs representing five functional groups (animal endosymbionts, endophytes, mycoparasites, plant pathogens, and saprotrophs). Based on relative abundance data, Basidiomycota were the most frequently detected phyla (50% of total sequences), followed by unidentified phyla, and Ascomycota. However, based on presence/absence data, the most OTU-rich phyla were Ascomycota (58% of total OTUs, 72 OTUs) followed by Basidiomycota and unidentified phyla. The majority of fungal OTUs were identified as saprotrophs (70% of successfully function-assigned OTUs) followed by plant pathogens. Finally, we used phylogenetic analysis based on the full-length ITS sequences to confirm the species identification of 14/36 OTUs with high bootstrap support (99–100%). Based on the numbers of sequence reads obtained per sample, which ranged from 3,047 to 13,463, we conclude that Pacbio sequencing can be a powerful tool for characterizing moderate- and possibly high-complexity fungal communities.

Identification of Microorganisms Dwelling on the 19th Century Lanna Mural Paintings from Northern Thailand Using Culture-Dependent and -Independent Approaches

“…These approaches are generally less laborious but require more efforts on molecular biology and bioinformatics. Culture-independent approaches have been rapidly developed over the past decade due to the applications of high-throughput sequencing technologies such as Next Generation Sequencing (NGS, e.g., 454 pyrosequencing, Illumina sequencing) and long-read Third Generation Sequencing (TGS, e.g., PacBio, Oxford Nanopore [ 38 , 39 , 40 ] Based on genetic and bioinformatic information obtained from culture-independent approaches, we can reasonably characterize the diversity and community composition as well as predict potential interactions and functions of microbes in various environments [ 41 , 42 , 43 ]. However, there are some pitfalls to culture-independent approaches, particularly relating to PCR biases [ 42 ].…”

Quantifying trade-offs in the choice of ribosomal barcoding markers for fungal amplicon sequencing: a case study on the grapevine trunk mycobiome

“…Indeed, above some threshold, increasing sequencing depth is not expected to yield more recovered diversity (27). Purahong et al (21) showed also that sequencing depth alone is a poor performance metric to evaluate the representation of the fungal community. Kennedy et al (27) suggested a threshold of 100 reads per sample beyond which additional reads are unlikely to substantially shift community assessment of environmental samples.…”

Long-Reads-Based Metagenomics in Clinical Diagnosis With a Special Focus on Fungal Infections

“…PacBio has also been used for metabarcoding analysis of environmental fungal samples using the shorter ITS2 minibarcode and the large ribosomal subunit (28S), which are often used by short-read barcode studies ( Cline and Zak, 2015 ; Kyaschenko et al, 2017 ). Sequencing of the full length ITS1/2 DNA barcoding region by PacBio sequencers has been demonstrated on a wide variety of fungal species ( James et al, 2016 ; Walder et al, 2017 ; Heeger et al, 2018 ; Tedersoo et al, 2018 ; Purahong et al, 2019 ; Tedersoo and Anslan, 2019 ). A comparison between PacBio and nanopore sequencers found PacBio to be more efficient in the metabarcoding of complex environmental fungal samples due to errors found in nanopore sequencing ( Loit et al, 2019 ).…”