Authors
James Robert White and W. Florian Fricke
Summary
Next-generation sequencing has been successfully used to characterize microbial communities based on the amplification and sequencing of phylogenetic marker genes, e.g. the 16S rRNA gene. In comparison to 16S rRNA amplicon sequencing-based procedures for bacterial and archaeal microbiota analysis, few comparable protocols have been made available to study fungal organisms. Here we describe the CloVR-ITS protocol for fungal microbiota analysis using internal transcript spacer (ITS) amplicon sequencing. CloVR-ITS includes well known bioinformatic tools for alpha and beta diversity analyses, suitable to process even large sequence datasets: A) QIIME 1 for sequence processing and beta phylogenetic analysis using different methods including UCLUST 2; B) UCHIME 3 for rapid identification of chimeric sequences; C) Mothur [4] for alpha diversity and ecological parameter calculations; D) BLASTN 5 for taxonomic sequence assignments using custom databases; E) Metastats [6] and custom R scripts for statistical and graphical evaluations. CloVR-ITS accepts as input either a single raw multiplex 454-pyrosequencer output file (i.e. pooled barcoded sequences from multiple samples), or alternatively, pre-processed sequences from multiple samples in separate files. CloVR-ITS is available as part of the CloVR package (http://clovr.org).
Further details
The protocol was published on Protocol Exchange on 14 February 2013. To see the entire protocol, click on the source link.
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