Create phylogenetic tree
The phylogenetic tree represents the relationship between the sequences in terms of the evolutionary distance from a common ancestor. In downstream analysis this tree is used for example in calculating the UniFrac distances.
Software: FastTree
An alternate option to most of the steps mentioned in phase 1 and phase 2 is to run IM-TORNADO (Jeraldo et al. 2014 in review). IM-TORNADO is an integrated pipeline that takes demultiplexed reads and trims low quality bases, does paired read stitching, removes chimeras and generates OTU table, phylogenetic tree and assigns taxonomy. Unique feature of IM-TORNADO is that it can analyze paired reads that do not overlap. Non-overlapping paired reads are typically generated from non-overlapping variable regions of 16S rRNA. Such studies try to utilize information in two variable regions instead of one variable region as in any standard 16S rRNA study to define OTUs.
Bibliography
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