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PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives
Author(s) -
Tedersoo Leho,
ToomingKlunderud Ave,
Anslan Sten
Publication year - 2018
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.14776
Subject(s) - amplicon , biology , barcode , internal transcribed spacer , computational biology , dna barcoding , genetics , phylogenetic tree , dna sequencing , indel , metagenomics , ribosomal rna , phylum , evolutionary biology , ion semiconductor sequencing , gene , polymerase chain reaction , computer science , single nucleotide polymorphism , genotype , operating system
Summary 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.