Table of Content

Instruction: How to use this workshop tutorial

Workshop Tutorial

• 1. Introduction (Introduction Slides)
• 2. Installation of the R software and packages for analyzing microbiome sequence data
• 3. Basic R operations
• 4. Use of the R DADA2 package to process the 16S rRNA gene sequence reads
  • Step 1. Create a working directory and download a set of demo Illumina 16S rRNA sequences
  • Step 2. Find the sequence data
  • Step 3. Quality plots
  • Step 4. Filter and trimm sequences
  • Step 5. Learn sequence error rates to build error models for denoise purpose
  • Step 6. De-replicate reads
  • Step 7. Denoise sequences based on the error model to product amplicon sequence variants (ASVs)
  • Step 8. Merge the pair-end reads to single reads
  • Step 9. Identify chimera and remove them
  • Step 10. Make a summary table for the above processes
  • Step 11. Assign ASVs with taxonomy
• 5. Use of the R Phyloseq package to study microbial diversity
  • Step 1. Load some necessary libraries
  • Step 2. Convert the ASV from DADA2 into a Phyloseq object
  • Step 3. Make some alpha-diversity plots
  • Step 4. Make some beta-diversity plots
  • Step 5. Plot genus and species level bar charts
  • Step 6. Identify differentially represented species
  • Step 7. Export tables for more downstream analysis using MicrobiomeAnalyst
• 6. Use of the MicrobiomeAnalyst online tools for statistical, visual and meta-analysis of microbiome data
• 7. Beyond microbiome sequence data – Meta-genomic and Meta-transcriptomic data

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