Targeted Gene Metagenomic Data Analysis    ◾    297

--m-metadata-file taxonomy/group_metadata.tsv \

--o-visualization taxonomy/groupedby-group-yoga-barplot.qzv

qiime tools view taxonomy/groupedby-group-yoga-barplot.qzv

7.3.6  Construction of Phylogenetic Tree

The phylogenetic tree is used to visualize the evolutionary relationship between sequences.

In the amplicon-based sequence data analysis, a phylogenetic tree is also required for gen-

erating some diversity indexes such as Faith’s phylogenetic diversity index and UniFrac

index.

QIIME2 provides several methods for constructing phylogenetic trees based on the

representative sequences generated from clustering or denoising. The methods include

alignment-based approaches for the de novo tree construction and a fragment-insertion

method that aligns features against a reference tree. It is recommended that the de novo

phylogenetic tree is constructed from long sequences only.

7.3.6.1  De Novo Phylogenetic Tree

In QIIME2, the de novo phylogenetic tree is constructed by using the following steps:

7.3.6.1.1  Multiple Sequence Alignment

The representative sequences obtained from clustering or denoising step are aligned using

“q2-align” plugin with “mafft” method. MAFFT is a program for multiple sequence align-

ment. For the tree files, we will create the “trees” subdirectory in the project directory.

mkdir trees

qiime alignment mafft \

--i-sequences dada2/rep-seqs_yoga_dada2.qza \

--o-alignment trees/aligned-rep-seqs_yoga_dada2.qza

7.3.6.1.2  Masking Sites of Low Information

The positions of low phylogenetic information on the sequences are masked to be avoided.

qiime alignment mask \

--i-alignment trees/aligned-rep-seqs_yoga_dada2.qza \

--o-masked-alignment trees/masked-aligned-rep-seqs_yoga_dada2.

qza

7.3.6.1.3  Creating a Tree

An unrooted tree is created from the aligned sequences using Fasttree that infers approxi-

mately maximum-likelihood unrooted phylogenetic trees from the sequence alignments.

qiime phylogeny fasttree \

--i-alignment trees/masked-aligned-rep-seqs_yoga_dada2.qza \

--o-tree trees/unrooted-denovoTr-tree.qza