PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives

Abstract

Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.

Boosting DNA metabarcoding for biomonitoring with phylogenetic estimation of operational taxonomic units’ ecological profiles

“…Obviously, longer DNA reads capture more historical signal. Hence, the quality of reads insertion is also expected to improve as read lengths produced by HTS will increase (Tedersoo, Tooming‐Klunderud, & Anslan, ). Finally, it should be noted that most of the species which were wrongly placed are marine (e.g., Guinardia striata , Stephanopyxis turris ) and therefore will not impede the computation of freshwater biotic indices like the IPS index.…”

Applications of sequencing technology in clinical microbial infection

“…The current is interfered when DNA or RNA molecules pass through it, causing a characteristic change in the current signal. In this process, the signal is analysed in real time to determine the base sequence of the DNA or RNA strand that is passing through the pore, which allows the entire DNA or RNA sequence to be determined …”

High-level classification of the Fungi and a tool for evolutionary ecological analyses

“…Many taxonomists place the unicellular Rozellomycota, Microsporidia and Aphelida within Fungi (James et al 2006a;Jones et al 2011a, Adl et al 2012James and Berbee 2012 and further studies on fungal classification), but other authors indicate the monophyly of Aphelida and Rozellomycota in a sister position to all other Fungi (Karpov et al 2013;2014b, 2017bLetcher et al 2013Letcher et al , 2017 and treat this socalled ARM clade as phylum Ophistosporidia (Karpov et al 2014b) or a part of the intentionally paraphyletic phylum Choanozoa, which includes protists at the base of Metazoa (Cavalier-Smith 2013; Ruggiero et al 2015). However, taxonomically more inclusive phylogenies place these groups separately-Rozellomycota and Microsporidia at the basal position of Fungi but Aphelida nested within 'chytrids' and/or zoopagaceous zygomycetes (Lazarus and James 2015;Tedersoo et al , 2018. Therefore, we suggest renaming of Aphelida to Aphelidiomycota to meet the standards of nomenclature.…”

“…The clades GS01 and Basal Clone Group 2 represent a potential successive sister lineage to all fungal phyla, albeit with limited statistical support (Tedersoo et al , 2018. Since nothing is known about the morphology of these clades, we consider these tentatively as subkingdom-level groups within Fungi, because of their supported monophyly with Fungi and divergence time of \ 1000 Ma.…”

“…The more readily alignable 18S and 28S rRNA genes tend to lack resolution at the level of species and functional groups by lumping ectomycorrhizal and saprotrophic fungal species in many cases. The alternative options include use of protein-encoding gene barcodes such as RPB2 (Vetrovsky et al 2016) or a long barcode spanning ITS and 18S or 28S (Timling et al 2014;Tedersoo et al , 2018. Mapping of OTUs to sequence-based phylogenies is difficult, because it essentially assumes building a backbone phylogeny that spans all genera of fungi and construction of multiple small trees associated to the backbone.…”