1. RDP – Capturing the Unclassified Use only on data that can be publicly shared. These are not secure tools.

2. Genboree RDP Output Tutorial 2 Dataset – QIIME chimeras removed – RDP Sample Period

3. Download files Raw.results.tar.gz

4. Unarchive and Decompress Use 7zip Seq.fna

5. Open in Bioedit

6. In Bioedit: – Ctrl +A – to select all sequences – Shift + Ctrl + C – to copy all sequence titles In Excel: – Paste into excel. In Column B (or other) – =left(a1,number_of_characters_in_titles) – Ctrl+Shift+Down arrow – Ctrl+D – to copy to all cells below Check your work. Select only your samples. Do not select blank cells. Copy the correct titles.

10. In Bioedit: Paste Over titles Save as: your_filename.fas In the pull down menu – choose fasta

11. rdp.cme.msu.edu

12. Make an Account

13. For very tiny datasets

14. very tiny datasets

15. Do not navigate away

16. For pyrosequenced datasets

20. You can navigate away and pick up the results later.

21. Check in while running?

22. Done: Download

23. What do you get back? Confidence file Classifications Failed classifications  Check this file. – Problems have happened if not empty. Hierarchy

24. Open classifications in excel Focus on Phylum for tutorial. Use any level.

25. Tutorial ease condense sample periods

26. Keep it Tidy Cut out what isn’t needed or being used.

27. Confidence in the Classification Sort on the confidence level Odd groups – Leave in or take out? Replace those below your confidence level Unclassified_ =concatenate($column$row,cell) $ keeps the column or row static in your formula as you drag to multiple cells

29. Copy to a new column Remove Duplicates

30. Even at the Phylum Level 60 categorical levels – (could be 2 for every known phylum)

31. To count by sample and phylum classification =countifs($K:$K,$O2,$A:$A,P$1) How to stop recalculation and manually restart – don’t crash your machine! You can easily cause hours of computation on large matrixes!

32. Stop Automatic Recalculation In the Options Menu Under Formulas F9

33. Fill Formulas and Check Cells

34. Copy Whole and Paste As Values

35. Sum Rows and Sort On (Your Favorite) Total is Customary Can rearrange as needed

36. Select Data and Titles Only

37. Make a 100% Stacked Chart Not very pretty

38. Switch Perspectives

39. Size Correctly

40. To Compare to Genboree RDP must be run png.result.tar.gz

41. What did we learn?

43. Some Problems Commonly Encountered Column formatting is not always followed with RDP output. To get a clean graph with all taxonomic levels on one column, you may need to sort and remove sections of data. Some have additional levels Some have fewer levels of classification

44. Additional Levels of Classification Delete Move over Delete Move over

45. Fewer Levels of Classification Common Trouble Makers Bacteroidetes Verrucomicrobia Acidobacteria Dehalococcoidetes Cyanobacteria Chloroplast Deltaproteobacteria OD1_genera_incertae_sedis TM7_genera_incertae_sedis Armatimonadetes WS3_genera_incertae_sedis Move Over