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 assignment. here, we utilized the third-generation pacific biosciences (pacbio) rsii sequel instruments to evaluate suitability of full-length internal transcribed spacer (its) barcodes longer rrna gene amplicons metabarcoding fungi, oomycetes other eukaryotes in soil samples. revealed multiple errors biases: taq polymerase substitution mis-incorporating indels sequencing homopolymers constitute major errors; sequence length biases occur during pcr, library preparation, loading instrument quality filtering; primer-template mismatches bias profile when using regular highly degenerate primers. platforms enable up 3000 bp, but remains slightly inferior illumina especially amplicons. full its barcode flanking small subunit greatly improve identification at phylum levels, respectively. conclude that pacbio provides a viable alternative organisms are relatively low diversity, require> 500-bp barcode for reliable identification or when phylogenetic approaches are intended.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.
Journal: The New phytologist
DOI: 10.1111/nph.14776
Year: 2018