How to‌ Use QIIME to Analyze 16S rRNA Gene Sequences from Microbial Communities Justin Kuczynski et al.
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Summary Basic Protocol 1: represents the first analysis steps typically performed on 16S DNA sequence data from microbial communities. Basic Protocol 1 consists of acquiring an example dataset, and assigning the DNA sequences in that study to the nine microbial communities included in the study. In typical usage, a researcher would substitute the data produced by a sequencing platform for the example data used here‌ Basic Protocol 2: consists of picking Operational Taxonomic Units (OTUs) based on sequence similarity within the reads, and picking a representative sequence from each OTU. The protocol also assigns taxonomic identities using reference databases, aligns the OTU sequences, creates a phylogenetic tree, and constructs an OTU table, representing the abundance of each OTU in each microbial sample. Basic Protocol 2 requires demultiplexed sequences such as those generated in the seqs.fna file from Basic Protocol 1.
Basic Protocol 3: consists of computing the within community diversity (alpha diversity) for each of the 9 microbial communities, and generating rarefaction curves (graphs of diversity versus sequencing depth). Basic Protocol 3 requires an OTU table and phylogenetic tree such as those produced in Basic Protocol 2. An OTU table heatmap showing taxonomy assignment for each OTU.
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