Genboree Microbiome Workbench 16S Workshop Part I March 11 th, 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: Workbench and Commons Differences Account – How to create your account? – ublic-commons?faq_id=493 ublic-commons?faq_id=493 Workshop Home – march march-2014

Workbench Where is it? Create a Group - Demo – Why? To serve as a project base – How to share it with others? – commons?faq_id=494 commons?faq_id=494 Create a Database - Demo – Why? To hold processed and pre-processed files – Using folders to organize the space – commons?faq_id=491 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 – commons?faq_id=492 commons?faq_id=492

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 

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

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 

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.

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 

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 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

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 

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 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 