Genboree Microbiome Workbench 16S Workshop Part I

Genboree Microbiome Workbench 16S Workshop Part I March 11th, 2014 Julia Cope Emily Hollister Kevin Riehle

Genboree Workflow • Create Group • Create Database • Create Project • Upload Files  • Create Samples (Sample Import using metadata file)  • Link Samples to Sequence Files (Sample File Linker)  • QC and Attach Sequences (Sequence Import)  • QIIME    • RDP 

Genboree • URL: http://www.genboree.org • Workbench and Commons Differences • Account • How to create your account? • http://genboree.org/theCommons/ezfaq/show/public-commons?faq_id=493 • Workshop Home • http://genboree.org/theCommons/projects/mw-march-2014

Workbench • Where is it? http://genboree.org/java-bin/workbench.jsp • Create a Group - Demo • Why? To serve as a project base • How to share it with others? • http://genboree.org/theCommons/ezfaq/show/public-commons?faq_id=494 • Create a Database - Demo • Why? To hold processed and pre-processed files • Using folders to organize the space • http://genboree.org/theCommons/ezfaq/show/public-commons?faq_id=491 • Create a Project - Demo • Why? To have a record of the major level processes that you have used on your data • Importance of tracking information for multiple users in a group • http://genboree.org/theCommons/ezfaq/show/public-commons?faq_id=492

Upload Files • What to import (upload) • Meta data • .sff (s) • Can both meta data and sffs be in one file? No - upload them separately. .sffs will need unpacking while meta data files will need converting. Shortcutting this step can cause odd problems down the line. • Importing files and choosing to extract will cause the system to queue the process. The process may take a few moments. • Now that I have it uploaded…How to edit and remove files? - Demo

Create Samples (Import) • Import samples singly or in multiples • Creating and adding samples to a set • Import Behavior • Assign samples to a set • What is a sample set? • Why use them? • Grouping for downstream analysis • Makes Genboree use faster on user (don’t have to move each file around) • Editing sample information

Create Samples (Import) • Import samples singly or in multiples: Demo • Creating and adding samples to a set • Input Window: Metadata file • Output Window: Target Database • Data> Samples & Sample Sets> Samples> Import Samples • Double check your Input, Target, and Settings • Import Behavior • Create New Record • Keep Existing • Merge and Update Use this one by default • Replace Existing • Assign Samples to new Sample Set • Name the folder or leave blank to not create a set • Can be added to a set later

Create Samples (Import) • What is a sample set? • Why use them? • Grouping for downstream analysis • Makes Genboree use faster on user (don’t have to move each file around) • Editing sample information • What isn’t possible (right now)? • Editing column titles • Adding single samples de novo

Sample Set Management • Demo. adding samples to a sample set • Input Window: Sample to be added • Output Window: Target Sample Set • Data> Samples & Sample Sets> Sample Sets> Add Sample to Sample Set • Demo. editing Sample (or Sample Set) data • Input Window: Sample to be edited • Output Window: Blank • Data> Samples & Sample Sets> Samples> Edit Samples • This is important for later stages • Makes Sequence Import easier and cleaner

Sample Set Management • Editing Sample (or Sample Set) data • Move boxes before saving or you will lose your edit.

Link Samples to Sequence Files • Sample file linker tool • The name is opposite the file positions required. • Arrangement in the Input Window: • .sff • Sample Set or • .sff • Sample • .sff • Sample • .sff • Sample • Output Window: Empty • Demo. how to do it and how to check it has been done.

Link Samples to Sequence Files • How to check your linked files? • The prompt screen on linking • The e-mail when complete • The Sample Edit tool – look for fileLocation column. • Demo. looking at linked fileLocation • Input Window: Sample to be edited • Output Window: Blank • Data> Samples & Sample Sets> Samples> Edit Samples

Sequence Import • Choose one or more samples to load sequences • Input Window: Sample(s) or Sample Set • Output Window: Target Database • Metagenome> Data Initialization> Import 16S rRNA Sequences • Check quality of import • Fixing the files when something has gone wrong • When it is possible? • When to start over? • Download files from Genboree

Sequence Import • Choose one or more samples to load sequences – Demo. • Input Window: Sample(s) or Sample Set • Output Window: Target Database • Metagenome> Data Initialization> Import 16S rRNA Sequences

Sequence Import • Check quality of import

Sequence Import • Fixing the files when something has gone wrong

Sequence Import • Fixing the files when something has gone wrong • When it is possible? • Bad barcode? • Sample info. wrong? • Primers • Region • Direction • Bad file? • When to start over?

Sequence Import • Download files from Genboree • Click on file • In Details Window, choose Download • Start with • sequences_metrics_ summary.xls • Easy to open • No compression

Sequence Import • When problems arise, check the: • sample.metadata – Does it match what you put in? • fasta.result.tar.gz – Look at the .fasta files • See barcodes • See primers • Notepad for metadata • Bioedit to open fasta • Use WINE on Mac

Genboree Microbiome Workbench 16S Workshop Part I