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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).

The idea is that there will be a lot of different workflows for analyses that we often carry out. These cover both marker gene (e.g. 16S, 18S, ITS as well as others) and metagenomic sequencing. Some parts of this are "core", but some parts will depend on what you want to get from your data and what your questions are. This is not a comprehensive list of all of the options that are out there!! These are some tools that we use the most, but there are many others out there - we encourage you to do your own research and decide which is the best for your purposes.

For example, for metagenome analyses, you may want to do:

• Initial pre-processing and QC (core)
• Taxonomic annotation with Kraken
• Checking coverage of obtained annotations

Or you may want to do:

• Initial pre-processing and QC (core)
• Annotation of reads with CARD RGI
• MAG assembly, binning, and curation with Anvi'o
• Annotation of contigs with CARD RGI

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

We also plan to make available both an "image" that has all current versions of programs pre-installed and is more-or-less ready to use, as well as instructions on setting up a computing environment for yourself.

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
• Other useful things like:
  • tmux
  • scp or other method for downloading from server

Using AWS instances

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

Tutorial data

• Download tutorial data including:
  • 16S short-read
  • 16S long-read
  • Metagenome short-read
  • Metagenome long-read

Amplicon workflows

• Basic Marker gene workflow in QIIME2
• Basic statistics and visualisation in QIIME2
• Functional prediction with PICRUSt2

Metagenomics initial pre-processing and QC

• Metagenomics initial Steps

Metagenomic taxonomic annotation

• Annotation of reads with Kraken2 (and taxonomic profiling with Bracken)
• Taxonomic profiling with MetaPhlAn
• Taxonomic profiling with Sylph
• Making Kraken2 and Bracken databases
• Checking coverage of obtained annotations
• Getting a tree for reads

Metagenomic functional annotation

• Annotation of reads with MMSeqs2
• Annotation of reads/contigs with CARD RGI
• Linking functions with taxonomy
• Functional profiling with 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

Full 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)