Genboree Microbiome Workbench 16S Workshop Part I

March 11th, 2014Julia Cope

Emily HollisterKevin 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/pu

blic-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

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 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

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