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Critical Issues in mycobiota analysis

Artikel i vetenskaplig tidskrift
Författare Bettina Halwachs
N Madhusudhan
R Krause
R. Henrik Nilsson
C Moissl-Eichinger
C Hoegenauer
GG Thallinger
G Gorkiewicz
Publicerad i Frontiers in Microbiology
Volym 8
ISSN 1664-302X
Publiceringsår 2017
Publicerad vid Institutionen för biologi och miljövetenskap
Språk en
Länkar journal.frontiersin.org/article/10....
https://doi.org/10.3389/fmicb.2017....
https://gup-server.ub.gu.se/v1/asse...
Ämnesord microbiota, mycobiota, internal transcribed spacer (ITS), 16S rRNA gene, multiple sequence alignment (MSA), OTU picking, formalin-fixed paraffin-embedded tissue (FFPE), DNA isolation
Ämneskategorier Biologiska vetenskaper, Mikrobiologi, Botanik, Genetik, Bioinformatik och systembiologi, Ekologi, Biologisk systematik, Infektionsmedicin, Mikrobiologi inom det medicinska området

Sammanfattning

Fungi constitute an important part of the human microbiota and they play a significant role for health and disease development. Advancements made in the culture-independent analysis of microbial communities have broadened our understanding of the mycobiota, however, microbiota analysis tools have been mainly developed for bacteria (e.g., targeting the 16S rRNA gene) and they often fall short if applied to fungal marker-gene based investigations (i.e., internal transcribed spacers, ITS). In the current paper we discuss all major steps of a fungal amplicon analysis starting with DNA extraction from specimens up to bioinformatics analyses of next-generation sequencing data. Specific points are discussed at each step and special emphasis is placed on the bioinformatics challenges emerging during operational taxonomic unit (OTU) picking, a critical step in mycobiota analysis. By using an in silico ITS1 mock community we demonstrate that standard analysis pipelines fall short if used with default settings showing erroneous fungal community representations. We highlight that switching OTU picking to a closed reference approach greatly enhances performance. Finally, recommendations are given on how to perform ITS based mycobiota analysis with the currently available measures.

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