MiSeq-Analysis

HTS Analysis Repository

A home for R, mothur, and some python based solutions for analysing your HTS datasets.

Tutorials/SOPs

Follow these standard workflows for a jumping off point.

• Aligning and Classifying Sequences using mothur (formerly FastQ to Mothur)
  • Start here. Learn how to use the command line program mothur to align and identify DNA sequences from several regions of 16S.
• Data Organization Throughout Your Project
  • Advice on setting up your metadata
  • Moving files to and from MSI
  • MSI workspace organization
  • R workspace organization
• Read Scaling, Normalization, Transformation, Rarefactions and Diversity
  • How to import your mothur output files into R for analysis
  • basics of phyloseq
  • OTU Richness, evenness, and coverage
  • The difference between proportional and relative abundance
  • Creating an even depth dataset
• Ordinations and Redundancy Analysis
  • Formerly known as beta diversity analysis
  • The differences between distance metrics
    • Bray Curtis, Jaccard, Manhattan, etc.
  • Mapping categorical and scalar values onto your microbial community
    • Principle components of analysis (PCA)
    • Principle coordinates of analysis (PCoA)
    • Non Metric Dimensional Scaling (NMDS)
  • Redundancy Analysis to connect environmental variables to your data
  • Minimum Spanning Trees for visualizing small differences
  • Cluster identification
• Describing Major Lineages and Differential Abundance
  • Who is in your samples?
  • Summarizing taxonomic differences at varying levels
  • Making stacked bar charts that aren’t lies
  • Visualizations with heat maps
  • Statistically measuring differences in abundance
  • Using ALDEx2 to detect significant differences
• FAPROTAX and Functions
  • What are the microbes doing?
  • Assign functions
  • Visualization of FAPROTAX results
  • Make your own function tables
• Source Sink Dynamics
  • Set up Sourcetracker with phyloseq data
  • Visualize sourcetracker results
  • Case study

General Help

• Installing Python
• SLURM computing (PDF)
  • Download metadata for this tutorial here.
  • This tutorial is slightly outdated. MSI has retired login nodes for both sftp and ssh access. Now connect directly to msi using ssh mesabi.msi.umn.edu -l yourx500 or ssh mangi.msi.umn.edu -l yourx500 and only login once!

Other Helpful Packages and Tutorials

• Having trouble in mothur? Check the SOP and the wiki
• Compositional analysis is complicated. The ALDEx2 package, used here frequently, is really helpful and the Gloor lab has extensive documentation and a helpful workshop
• I’m a big fan of mixOmics. For fancier PCA options, check it out.

Helpful Publications

• Miranda’s long list of favorite bioinformatics articles will someday become available.

Authors

Miranda Galey @mgaley-004

Contact at galey004-at-umn.edu

Learn more about my work