1.构建系统发育树

mkdir phylogeny
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences filtered_rep_seqs/final_rep_seqs.qza \
--o-alignment filtered_rep_seqs/final_rep_seqs_aligned.qza \
--o-masked-alignment filtered_rep_seqs/final_rep_seqs_masked.qza \
--o-tree phylogeny/unrooted-tree.qza \
 --o-rooted-tree phylogeny/rooted-tree.qza

2.稀释曲线

mkdir rarefaction
qiime diversity alpha-rarefaction \
--i-table filtered_table/final_table.qza \
--i-phylogeny phylogeny/rooted-tree.qza \
--p-max-depth 59078 \
--m-metadata-file sample-metadata.tsv \
--o-visualization rarefaction/p-max-depth-59078-alpha-rarefaction.qzv

3.多样性分析
1）根据ASV的最小测序深度设置sampling参数

mkdir diversity
qiime diversity core-metrics-phylogenetic \
--i-phylogeny phylogeny/rooted-tree.qza \
--i-table filtered_table/final_table.qza \
--p-sampling-depth 29853 \
--m-metadata-file sample-metadata.tsv \
--output-dir diversity/sample-depth-29853-core-metrics-results

2)alpha多样性分析-faith_pd

qiime diversity alpha-group-significance \
--i-alpha-diversity diversity/sample-depth-29853-core-metrics-results/faith_pd_vector.qza \
--m-metadata-file sample-metadata.tsv \
--o-visualization diversity/sample-depth-29853-core-metrics-results/faith-pd-group-significance.qzv

3)alpha多样性分析-shannon

qiime diversity alpha-group-significance \
--i-alpha-diversity diversity/sample-depth-29853-core-metrics-results/shannon_vector.qza \
--m-metadata-file sample-metadata.tsv \
--o-visualization diversity/shannon_compare_groups.qzv

4）beta多样性分析

qiime diversity beta-group-significance \
--i-distance-matrix diversity/sample-depth-29853-core-metrics-results/unweighted_unifrac_distance_matrix.qza \
--m-metadata-file sample-metadata.tsv \
--m-metadata-column Groups \
--o-visualization diversity/unweighted-unifrac-subject-significance.qzv

5）三维PCOA

qiime emperor plot \
--i-pcoa diversity/sample-depth-29853-core-metrics-results/unweighted_unifrac_pcoa_results.qza \
--m-metadata-file sample-metadata.tsv \
--o-visualization diversity/unweighted-unifrac-emperor-height.qzv

4.物种组成柱状图

mkdir barplot
qiime taxa barplot \
--i-table filtered_table/final_table.qza \
--i-taxonomy taxonomy/final_taxonomy_sliva.qza \
--m-metadata-file sample-metadata.tsv \
--o-visualization barplot/final_taxa_barplots_sliva.qzv