Microbiome Hub  
UW Biotechnology Center Bioinformatics Resource Center  
Please direct questions to: brc@biotech.wisc.edu

Summary

PI Name Name
Project Description Qiime2 Analysis
Number of samples 34
Number of features 759
Total frequency 263878
Report Generation Date 2019-04-11

Taxa Summary

The relative abundance for each level of taxa L2(Class) to L7(Species) can be found in the “taxa_summary” folder. To view full interactive Taxa Summary drag and drop file - taxa-bar-plots.qzv in to https://view.qiime2.org/
The barplot below shows the 10 most abundant bacterial taxa at genus level.

Alpha Rarefaction

(documentation)

Interactive rarefaction curve can be found in alpha_rare folder based on the treatments and individual samples.

Beta Diversity

(documentation)
Interactive beta diversity plots can be found in diversity folder. Files with *emperor.qzv can be uploded to https://view.qiime2.org/ to visualize PCoA plots. This PCoA plot is created with bray curtis diversity metrics and also provides P values for pairwise comparison.

Heatmap

This heatmap represents the 30 most abundant bacteria taxa at Genus level.

Phylogeny

The ASV/OTU phylogeny tree can be found in the phylogeny folder in the file rep_seqs_filt_aligned_masked_tree_rooted.qza and can be directly uploaded into iTOL webstie



Software

QIIME Version 2019.1.0
Denoising Dada2
Alignment PyNAST
Reference Database 16S-Silva-132
Taxonmy Assignment classify-sklearn
Phylogeny Generation FastTree

Workflow Chart









Microbiome Analysis was performed by the UW biotechnology center using Quantitative Insights Into Microbial Ecology (QIIME2)[1] version 2. Illumina sequencing reads will be denoised and quality filtered using the denoising program DADA2. This will trim low quality bases, filter out noisy sequences, correct errors in marginal sequences, remove chimeric sequences and singletons, join denoised paired-end reads and then dereplicate those sequences. The resultant dereplicated sequences are termed as “Amplicon sequence variant (ASV)” and is equivalent to operational taxonomic unit (OTU). Sequence variants will be aligned and masked using Mafft and the phylogenetic tree of the ASV’s created using FastTree. Taxanomy will be assigned using Bayesian classifier based on a pretrained Silva database curated to the exact 16s amplicon region. The resulting biom formatted table describing the occurrence of bacterial phylotypes within each sample will be generated for further downstream analysis. Low frequency reads (<0.01%) will be filtered from the Biom-formatted table. Alpha rarefaction curves using Shannon, Chao1, Simpson and observed were calculated for all samples with a rarefaction upper limit of median depth/sample count and the alpha diversity between different treatments will be compared using Kruskal Wallis test. Samples were removed from further characterization if they did not contain sufficient reads. Beta diversity was calculated and ordination plots were generated using Bray-Curtis and Jaccard (Non-Phylogenetic), Weighted and unweighted Unifrac (Phylogenetic) on ASV data leveled according to the lowest sample depth.

Please acknowledge BRC in your manuscript or presentation. If you think our analysis contributes to your research intellectually please consider authorship for our bioinformaticians.