Home ‐ Microbiome Helper 2

We have been continually been updating our SOPs and workflows, and as they are now substantially different than they were at the time of publication and release of Microbiome Helper (2017), we will be working on updating our scripts, SOPs, workflows and tutorials for a second release of Microbiome Helper (i.e., Microbiome Helper 2).

We will update this page as we work on these different aspects.

In this, we plan to have:

General notes on analysis

• Things you need before starting
• Computational resources required
• Setting up environments for analysis/installation of all programs

Introduction to Unix

• Brief introduction to the command line

Using AWS instances

• Introduction to Amazon instances and what they are
• Setting up an AWS instance for your analysis

Tutorial data

• 16S/amplicon Illumina
• 16S/amplicon PacBio
• Metagenome Illumina
• Metagenome PacBio

Amplicon SOPs

• Amplicon SOP (Illumina and PacBio)
• Basic statistics and visualisation
• Functional prediction with PICRUSt2

Metagenomics initial pre-processing and QC

• Metagenomics Standard Operating Procedure Initial Steps

Metagenomic taxonomic annotation

• Annotation of short reads with Kraken2 and Bracken
• Annotation of long reads with Kraken2 (or other?)
• Annotation with MetaPhlAn (short or long reads)
• Making Kraken2 and Bracken databases
• Checking coverage of obtained annotations
• Getting a tree for reads

Metagenomic functional annotation

• MMSeqs2
• CARD RGI
• Linking this with taxonomy
• HUMAnN
• Obtaining annotations for gene of interest with HMM, including:
  • Making HMMs of gene of interest
  • Running HMMs with single copy genes
  • Normalising output to gene lengths

MAGs

• MAG assembly, binning, and curation with Anvi'o

Analysis workflow

• Importing data:
  • 16S
  • Metagenomics taxonomic data
  • Functional data (predictions or metagenomic annotations)
  • Stratified data
• Overview of samples:
  • Basic stacked bar plots
  • Heatmaps
  • Phylogenetic trees
  • Including fundamentals of adding plots together? (e.g. ordering by taxa so that a heatmap will appear in the same order as the tree and can be linked?)
• Alpha/beta diversity:
  • Alpha diversity metrics and visualisation
  • Beta diversity metrics and visualisation - ordination vs others e.g. heatmap or dendrogram
  • Look at taxonomic contributions to function
• Differential abundance:
  • ANCOM
  • ALDEx
  • MaAsLin
  • Corncob
  • radEmu
• Longitudinal analysis
• Output R Notebook
• Combining taxonomic/functional visualisations with JarrVis

Other useful things

• Downloading reads from SRA
• Uploading reads to SRA (or ENA)