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

Castaño Soler, Carles and Berlin, Anna and Brandström Durling, Mikael and Ihrmark, Katarina and Lindahl, Björn and Stenlid, Jan and Engelbrecht Clemmensen, Karina and Olson, Åke (2020). Optimized metabarcoding with Pacific biosciences enables semi-quantitative analysis of fungal communities. New Phytologist. 228 , 1149-1158
[Research article]

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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 related to GC content were observed. Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.

Authors/Creators:Castaño Soler, Carles and Berlin, Anna and Brandström Durling, Mikael and Ihrmark, Katarina and Lindahl, Björn and Stenlid, Jan and Engelbrecht Clemmensen, Karina and Olson, Åke
Title:Optimized metabarcoding with Pacific biosciences enables semi-quantitative analysis of fungal communities
Series Name/Journal:New Phytologist
Year of publishing :2020
Volume:228
Page range:1149-1158
Number of Pages:10
Publisher:WILEY
ISSN:0028-646X
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution-Noncommercial 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Microbiology (Microbiology in the medical area to be 30109)
(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Genetics (medical genetics to be 30107 and agricultural genetics to be 40402)
Keywords:high-throughput sequencing, Illumina MiSeq, ITS, metabarcoding, metabarcoding biases, mock community, PacBio
URN:NBN:urn:nbn:se:slu:epsilon-p-107178
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-107178
Additional ID:
Type of IDID
DOI10.1111/nph.16731
Web of Science (WoS)000549084900001
ID Code:22681
Faculty:NJ - Fakulteten för naturresurser och jordbruksvetenskap
S - Faculty of Forest Sciences
Department:(NL, NJ) > Dept. of Forest Mycology and Plant Pathology
(S) > Dept. of Forest Mycology and Plant Pathology

(NL, NJ) > Dept. of Soil and Environment
(S) > Dept. of Soil and Environment
Deposited By: SLUpub Connector
Deposited On:01 Mar 2021 10:43
Metadata Last Modified:01 Mar 2021 10:51

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