Optimized metabarcoding with Pacific biosciences enables semi-quantitative analysis of fungal communities.

The New Phytologist
Carles CastañoÅke Olson

Abstract

Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases relat...Continue Reading

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Citations

Feb 20, 2021·Parasites & Vectors·Peter Halvarsson, Johan Höglund
Mar 24, 2021·Ecology Letters·Karina Engelbrecht ClemmensenBjörn D Lindahl
May 3, 2021·Ecology Letters·Björn D LindahlJohan Stendahl
Aug 21, 2021·Scientific Data·Mariana KlugeSari Peura

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