By Angus Ball

Introduction

Welcome to the dry lab procedures page! I'm doing my best to make things logical, organized and helpful for people other than me, BUT I'm also writing these S.O.Ps as I need them/when things go wrong. So to get a complete data analysis package theres gonna be some wibbly wobbly timey wimey stuff that needs to happen. So while i go through datasets and things go wrong i'll try to keep this list updated so you can see where things go wrong and links to pages where I explain how to fix them.

You can follow this tutorial page through, and I'll regularly give papers to read or if you feel like getting ahead heres a bit of a reading list:

Bioinformatics reading list

Read This detailed(ish) introduction to the systematic problems of NGS sequencing and deciding on your primers ...or else

• Compositionality and You

Giving data to genome Quebec

1. So you just got some data? Getting data off Genome Quebec

Starting from raw data

1. You think your computer has the beans to do what needs to be done? Maybe it does, but if you can't be asrsed waiting a day for the program to run time to get on unbc's high performance computer (Genuinely you can do it on your home computer, but have a talk with me (angus) to see if you want to get on the hpc or just suffer for a day)
   • The High Profile Computer at UNBC
   • Then use DADA2 to process your raw data...
   • DADA2

Processed data and Data analysis

Sweet you completed DADA2 and all it entails! Time for data analysis.

1. First you must make a phyloseq object Making a Phyloseq Object, this is how your data is "held" for data analysis programs

   • Start by a quick and dirty introduction into how to import data into R and what your data should look like
   • Importing data into R
   • then go make your phyloseq object
   • Making a Phyloseq Object
   • Hey do you like Fun and or guilds? if you want to use FUNGuild or FungalTraits go read how to make a phyloseq object and then go here
     • FungalTraits importing Data
     • FUNGuild importing Data NOTE: FUNGuild, however fun, is deprecated, use Fungaltraits
   • Hey do you like Fun and or guilds? But HATE fungi? What if you wanted to assign functional genes to bacterial sequences? you'd need to use PICRUSt2
     • I haven't written the tutorial for this yet but it looks COMPLICATED so maybe run some of the other analyzes first to get a good grip on R first
     • PICRUSt2

2. Sweet! After making a phyloseq object go make a relative abundance graph of a high level domain like kingdom or phylum. Do NOT hide samples under <1% abundance.

   • Relative Abundance Graphs
   • Was there stuff in there that shouldnt be? I know I had bug samples in my fungal DNA, you might also have plant DNA in your 16S sequences. Decide if you really want those samples/taxa to exist. If not go here
     • Removing samples and taxa and saving your phyloseq object

3. Now you can make a relative abundance graph for real with all the bells and whistles you like!

   • Relative Abundance Graphs
   • (Its the same tutorial as earlier)

4. With your data cleaned its time for some analysis

   • First read this as a pretense to metabarcoding data analysis
   • Then you can start doing some analysis
   • Alpha Diversity measurements and tests
   • Beta Diversity
   • Differential abundance
   • Network analysis