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

2. Genboree 16S Workshop Learning Objectives – Students should be able to take.sff files and user supplied information and produce: Metadata File PCoA Classification Distribution Expectations – Apply topics learned today before next meeting – Be able to discuss where issues arise – Be able to move knowledgeably through the whole Genboree Workflow

3. Genboree 16S Workshop Part II Learning Outcomes – Newer database version of RDP – How to take advantage? – Students should take user.sff files and user created metadata file and produce: (I can provide files if needed.) PCoA (QIIME) Classification Distribution (RDP) Expectations – Apply topics learned in tutorial – Be able to discuss where in the process issues arose – Have a hypothesis about your data issues if they happen

4. Workshop Outline 16S Metadata File Genboree Workbench Workflow – Account – Group – Database – Project – Loading your files/samples/sequences (and linking) – QIIME – RDP – How to get help Wrap Up and Preparation for 2 nd Installment

5. Resources Genboree Home Screen – http://genboree.org http://genboree.org Tutorials are located in the Genboree Commons – You must be signed in to open the following link – http://genboree.org/theCommons/projects/mw-march-2014 http://genboree.org/theCommons/projects/mw-march-2014 – Tutorial 1 Data Set: http://www.genboree.org/microbiome/include/data/tutorial_sequen ce_file.sff.gz http://www.genboree.org/microbiome/include/data/tutorial_sequen ce_file.sff.gz – Tutorial 2 Data Set: http://genboree.org/theCommons/attachments/3545/Tutorial_2.zip Projects are accessed through the Genboree Workbench

7. 16S What is it? What part is being sequenced? – Here? – Elsewhere? How is this accomplished? – DNA to bead to light – Intro. to flow data and.sff file content – OUTPUT is an.sff file – Aside on zipping methods and large file transfers

8. Allmetrics.net Sales Material Tortoli E Clin. Microbiol. Rev. 2003;16:319-354 What is it? 16Svedberg (small sub-unit of the ribosome) What part is being sequenced? Here? - TCMC sequences the V5-V3 by 454 Elsewhere? - V3-V5, V1-V3, V9, V7-V9…many more. Know your variable regions 16S

9. How is this accomplished? – DNA to bead to light http://cage.unl.edu/equipmentsoftware.shtml 454 Life Sciences Sales Materials

10. 16S How is this accomplished? – DNA to bead to light http://cage.unl.edu/equipmentsoftware.shtml 454 Life Sciences Sales Materials

11. 16S How is this accomplished? – DNA to bead to light – Intro to flow data and sff file content – OUTPUT is an.sff file – Standard Flowgram Format All reads are structured as linker-tag-primer Provides both identity and quality information http://cage.unl.edu/equipmentsoftware.shtml Allmetrics.net Sales Material

12. Genboree Workflow Take one step back from the Genboree Workflow and talk about input files. What do you do with your files? From: Genboree.org help files Meta- data.sff

13. Genboree Workflow What do you do with many files? Genboree takes.zip,.gzip,.txt, and.sff files – Compressed files are easier and faster to move – Multiple files are easier to move when compressed together in an archive Meta- data.sff.sff(s) should be archived and compressed. Meta data files are very small and do not need compression. Meta- data

14. Metadata Files What data must you have? How should it be formatted for Genboree? What can you include? How to make it tab-delimited Include variable region or primer? Directional awareness on primers

15. Metadata Files What data must you have? – name – barcode – region or proximal & distal – First column must begin with # – #No_spaces_are_allowed_in_column_names_0123456789 How should it be formatted for Genboree? – Tab delimited What can you include? How to make it tab-delimited? Include variable region or primer? Directional awareness on primers

16. Metadata Files How to determine which to include - variable region or primers Directional awareness on primers Demo of making and saving as tab delimited #namebarcodeproximaldistalregionbody_site S_700033665CCGTTCCTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700035861ACCGGCGTTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700095543ACGAATTAACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700095850AACCGGATACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700101600AACGGAACGCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool T_700016994AATAACCGTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700095565TTAATGGAACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700095872CGGACCGGAACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700101388CCGAACGACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700101622TTCGTTCTTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat or

17. #namebarcodeproximaldistalregionbody_site S_700033665CCGTTCCTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700035861ACCGGCGTTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700095543ACGAATTAACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700095850AACCGGATACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700101600AACGGAACGCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool T_700016994AATAACCGTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700095565TTAATGGAACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700095872CGGACCGGAACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700101388CCGAACGACCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat T_700101622TTCGTTCTTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Throat Metadata Files - Demo Select the data above and Copy. Paste into Excel or an open source spreadsheet program. Be sure all entries are free of spaces and special characters and that all samples have the same number of columns. Avoid the column titles "state" and "type". Save As and select tab-delimited. Name your file in a clear and consistent manner. or

18. Metadata Files How to determine variable region vs. primer inclusion Directional awareness of primers If you aren’t sure, ask! What are these files often called: mapping, metadata, oligos, or linker-primer file. (Many others possible.) #namebarcodeproximaldistalregionbody_site S_700033665CCGTTCCTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool S_700035861ACCGGCGTTCCCGTCAATTCMTTTRAGTCTGCTGCCTCCCGTAGGV3V5Stool Allmetrics.net Sales Material

19. Metadata Files Another example: Tutorial Set 2 Metadata What possible issues may arise with this metadata file? sampleNametagproximaldistalregionsample_periodtype Ferm_5AGCTTCGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V35Fermentation Ferm_2GCCATACATTGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V32Fermentation Ferm_3GCCAGCAAGTGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V33Fermentation Ferm_4CGTTAAGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V34Fermentation Ferm_1CTAACAGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V31Fermentation Soil_1ACGCAAAAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V31Soil Soil_2CTAACTAAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V32Soil Soil_3GCGACCTAGTGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V33Soil Soil_4AAGAATCAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V34Soil Soil_5AGCGCAGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V35Soil

20. Metadata Files Another example What possible issues may arise with this metadata file? Change name => #name (or any #1 st entry) Change tag => barcode Change type => sample_type (do not name columns ‘type’ or ‘state’) Demo. making and saving as tab-delimited #namebarcode proximaldistalregionsample_period sample_type Ferm_5AGCTTCGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V35Fermentation Ferm_2GCCATACATTGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V32Fermentation Ferm_3GCCAGCAAGTGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V33Fermentation Ferm_4CGTTAAGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V34Fermentation Ferm_1CTAACAGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V31Fermentation Soil_1ACGCAAAAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V31Soil Soil_2CTAACTAAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V32Soil Soil_3GCGACCTAGTGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V33Soil Soil_4AAGAATCAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V34Soil Soil_5AGCGCAGAGAGTTTGATCNTGGCTCAGCAGCMGCCGCNGTAANACV1V35Soil

21. 7zip Zipping methods and large file transfers Compression and archiving of files Uncompressing in an easy to use format for PCs Demo compressing –.sff (s) – http://www.7-zip.org/ http://www.7-zip.org/ From: 7-zip.org

22. 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 