RDP – Capturing the Unclassified

Use only on data that can be publicly shared.

These are not secure tools.

Genboree RDP Output

• Tutorial 2 Dataset– QIIME• chimeras removed

– RDP• Sample Period

Download files

• Raw.results.tar.gz

Unarchive and Decompress

• Use 7zip• Seq.fna

• Open in Bioedit

• 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.

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

rdp.cme.msu.edu

Make an Account

For very tiny datasets

very tiny datasets

very tiny datasets

• Do not navigate away

For pyrosequenced datasets

You can navigate away and pick up the results later.

Check in while running?

Done: Download

What do you get back?

• Confidence file• Classifications• Failed classifications Check this file. – Problems have happened if not empty.

• Hierarchy

Open classifications in excel

• Focus on Phylum for tutorial. Use any level.

Tutorial ease condense sample periods

Keep it Tidy

• Cut out what isn’t needed or being used.

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

Copy to a new columnRemove Duplicates

Even at the Phylum Level

• 60 categorical levels – (could be 2 for every known phylum)

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!

Stop Automatic Recalculation

• In the Options Menu• Under Formulas• F9

Fill Formulas and Check Cells

Copy Whole and Paste As Values

Sum Rows and Sort On (Your Favorite)

• Total is Customary• Can rearrange as needed

Select Data and Titles Only

Make a 100% Stacked Chart

• Not very pretty

Switch Perspectives

Size Correctly

To Compare to Genboree

• RDP must be run• png.result.tar.gz

What did we learn?

1 2 3 4 50%

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100%TenericutesSynergistetesOD1FusobacteriaDeinococcus-ThermusBRC1ArmatimonadetesThermotogaeOP11GemmatimonadetesTM7LentisphaeraeWS3DeferribacteresNitrospiraSpirochaetesActinobacteriaVerrucomicrobiaPlanctomycetesCyanobacteria/ChloroplastChlorobiAcidobacteriaBacteroidetesChloroflexiUnclassified_othersUnclassified_ChloroflexiUnclassified_ProteobacteriaUnclassified_FirmicutesProteobacteriaFirmicutes

What did we learn?

1 2 3 4 50%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%TenericutesSynergistetesOD1FusobacteriaDeinococcus-ThermusBRC1ArmatimonadetesThermotogaeOP11GemmatimonadetesTM7LentisphaeraeWS3DeferribacteresNitrospiraSpirochaetesActinobacteriaVerrucomicrobiaPlanctomycetesCyanobacteria/ChloroplastChlorobiAcidobacteriaBacteroidetesChloroflexiUnclassified_othersUnclassified_ChloroflexiUnclassified_ProteobacteriaUnclassified_FirmicutesProteobacteriaFirmicutes

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

Additional Levels of Classification

Delete Move over Delete Move over

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