[Using *Greengenes* to Classify Bacterial DNA Sequences]{.underline}

**[Introduction]{.underline}**

As people, we categorize the world around us. You can tell a sports car
from an SUV from a motorcycle, all vehicles. You know which clothes get
hung in your closet and which go into your dresser, and into which
drawer. Any three year old can tell the difference between a turtle and
a puppy dog. They may not be able to tell you exactly what
differentiates them, but they will probably be able to give you some
characteristics.

Biologists study and classify living things. We categorize life based on
a number of different characteristics. The main divisions, called
kingdoms, are separated based upon a very few, but distinct
characteristics, such as whether they obtain energy from the sun or
another living thing. Each kingdom is further broken down into different
phyla, having fewer organisms but more in common. This categorization
continues down to the species level.

Before advancements in technology, specifically in microbiology and
biochemistry, biologists used observable characteristics to categorize
living things. Today, however, biologists consider the comparison of
organisms' DNA to be the most accurate means of classification. In this
activity you will do just that, use sequences of DNA to classify a group
of prokaryotic microbes. The DNA sequence we are using is the gene for
the small subunit of the ribosome. You will learn more about this gene
during the lesson.

You are to write an introduction for this activity. It should be typed,
double spaced and will include answers to the following questions:

1.  What is the defining characteristic which separates a prokaryote
    from a eukaryote? What are the two kingdoms of prokaryotes?

2.  What are the similarities and differences between DNA & RNA?

3.  What are the three types of RNA and what does each do?

**[Objective]{.underline}**:

A.  [Overall]{.underline}:

> Students will work in pairs, and using a sample set of DNA sequences,
> will classify them and create a tree to represent their relative
> evolutionary closeness.

B.  [Day 1]{.underline}:

> Students will:
>
> 1.become familiar with *Greengenes* and the Tutorial.
>
> 2\. use the Tutorial to learn more about the 16S rRNA gene.
>
> 3\. use the Tutorial to review mutations.
>
> 4\. use the Tutorial to learn about near neighbors and type strains.
>
> 5\. use *Greengenes* to align DNA sequences to the database.

C.  [Day 2]{.underline}:

> Students will:
>
> 1\. review aligned sequences and compare with unaligned
>
> 2\. use the Tutorial to learn about chimeric sequences.
>
> 3\. use *Greengenes*/Bellerophon to remove chimeric sequences.

D.  [Day 3]{.underline}:

> Students will:
>
> 1\. use the Tutorial to learn about taxonomies.
>
> 2\. use *Greengenes* to classify the sequences.

E.  [Day 4]{.underline}

> Students will:
>
> 1\. Review the classifications of submitted sequences.
>
> 2\. Compare taxonomies by looking at multiple classifications of the
> same sequence.

**[Materials]{.underline}**:

Desktop or laptop computers (1 for every 2 students)

high speed internet connection

*Greengenes* ([http://greengenes.lbl.gov](http://greengenes.lbl.gov/))

Mega (opensource program for creating phylogenetic trees)

Text and spreadsheet programs, (preferably Word & Excel) on computers

Networked printer

**[Procedures]{.underline}**:

**Day 1:**

1)  **[To begin:]{.underline}**

    i.  Set up with a partner at a computer

    ii. Launch a web browser (Firefox, Safari, Internet Explorer)

    iii. Open the following web site:
         [http://greengenes.lbl.gov](http://greengenes.lbl.gov/)

    iv. Read through the text at the top of the page to gain an overall
        idea about what *Greengenes* is designed to do and answer
        question #1 in Part 1 of the Conclusion.

    v.  On the left hand side there is a menu box titled "About". In
        that box, click on "Tutorial".

    vi. Before you proceed with reading the tutorial, first create a
        split screen between the *Greengenes* and Tutorial pages (for
        **PC** users). To do this:

        a.  On the bottom of your screen you can see the windows that
            are active on your desktop. The Tutorial page should be the
            highlighted one.

        b.  Hold down the Ctrl key and click on the *Greengenes* page.

        c.  Now, with both highlighted, right click on one of them and
            select "Tile Vertically". This will split your screen
            between the two pages.

> (**Mac** users can manually resize the windows or use the F9 key to
> bounce between the *Greengenes* and Tutorial.)

vii. Please read through the Tutorial homepage. It will give you an idea
     as to what it is intended to cover, the files you can expect to use
     with and receive from *Greengenes*, as well as background
     information.

     1.  In the introductory paragraph is a hyperlink for the term
         "FASTA". Click & read. In that definition, click on the "learn
         more" link and read.

     2.  Once you have read about FASTA files, answer questions 2 & 3 in
         Part 1 of the Conclusion.

     3.  Underneath the table on the Tutorial main page is a link to
         learn more about the 16S rRNA gene. Click and read. Then answer
         questions 4 & 5 in Part 1 of the Conclusion.

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2)  **[Using *Greengenes* with the Tutorial]{.underline}**:

    i.  On the left is a menu bar called "Tutorial Topics". Click on
        "Sample Data" to open that page.

    ii. On this page you will see a list of the files. You are going to
        start with the first one, "Sample Data FASTA". Before you begin,
        to get an idea of what it is that *Greengenes* is doing, double
        click on the first file, Sample Data FASTA. This is what a real
        FASTA file looks like, long strings of nucleotide bases
        separated by unique identification names for each. Those long
        strings of nucleotide bases are the **actual** DNA codes for the
        16S rRNA gene of different prokaryotic organisms.

    iii. Once you have looked at the file please close it. You will not
         need it open at any point for the rest of this lesson.

    iv. Please read the directions on the Sample Data page for saving
        the Unaligned Sample file locally. For our purposes the
        **desktop** will be good place to save.

3)  **[Aligning Sequences]{.underline}**:

    i.  Navigate to the Tutorial home page. Look at the chart that shows
        inputs and outputs. You already have a FASTA file, so we will be
        starting in the second column. Feel free to investigate the
        first column to see how get a FASTA file, but again, you already
        have one.

    ii. Click on the second column, "Aligning Your Report".

    iii. In the second paragraph of the Alignment page is a link to a
         review of mutations. Click and read. Then, answer questions 6-8
         in Part 1 of the Conclusion.

    iv. At the top of the *Greengenes* page you will see a series of
        buttons. Click on the one that says "Align".

    v.  Work through the steps for alignment on the *Greengenes* page
        using the Tutorial to assist you as needed. For each bold
        heading on the *Greengenes* page there is a corresponding page
        in the Tutorial to explain it.

        1.  In step three of Alignment in the Tutorial is a link to a
            page that explains near neighbors. Click and read.

        2.  Once you have read, answer question 9 in Part 1 of the
            Conclusion.

    vi. To make sure that your aligned sequences are sent to a valid and
        accessible email address, **use** **your** **school email
        address**.

    vii. We will continue with the next step tomorrow, checking for
         chimeric sequences. For now, once you have submitted your FASTA
         file to be aligned, do the following:

         1.  Quit the browser you were using (Internet Explorer,
             Firefox, etc.)

         2.  Find the icon for the Sample Data FASTA and drag it to the
             trash.

         3.  Shut down the laptop completely -- **WAIT** for the screen
             to go completely black before you close the top!!

         4.  Return the laptop to the cart and check out with the
             teacher.

**Day 2:**

> [Objectives, Day 2]{.underline}:
>
> Students will:
>
> 1\. review aligned sequences and compare with unaligned
>
> 2\. use the Tutorial to learn about chimeric sequences.
>
> 3\. use *Greengenes*/Bellerophon to remove chimeric sequences.

4)  **[To begin: - (a review)]{.underline}**

    i.  Set up with a partner at a computer

    ii. Launch a web browser (Firefox, Safari, Internet Explorer)

    iii. Open the following web site:
         [http://greengenes.lbl.gov](http://greengenes.lbl.gov/)

    iv. On the left hand side there is a menu box titled "About". In
        that box, click on "Tutorial".

    v.  Before you proceed with reading today's lesson, first create a
        split screen between the *Greengenes* and Tutorial pages (for
        **PC** users). To do this:

        a.  On the bottom of your screen you can see the windows that
            are active on your desktop. The Tutorial page should be the
            highlighted one.

        b.  Hold down the Ctrl key and click on the *Greengenes* page.

        c.  Now, with both highlighted, right click on one of them and
            select "Tile Vertically". This will split your screen
            between the two pages.

> (**Mac** users can manually resize the windows or use the F9 key to
> bounce between the Greengenes and Tutorial.)

5)  **[Chimeric Check]{.underline}**:

    a.  Before you begin the check for chimeras (we'll get to what they
        are in just a moment!!), let's see what the alignment tool did
        to the sequences you submitted.

        i.  Open your email and find the email returned to you by
            *Greengenes*.

        ii. It will have two files attached. One is an aligned FASTA
            file. The other is a spreadsheet explaining the outcome of
            the alignment process on your submitted sequences. Open the
            aligned FASTA file.

        iii. Next, navigate to the sample data page on the Tutorial
             page. Double click on "Sample Data FASTA". Look at this
             unaligned file. Compare it with the aligned file returned
             to you by *Greengenes*. The areas dense with bases are the
             conserved regions of the gene; the areas dense with
             dashes/spaces are the hyper variable regions of the gene.

        iv. Answer questions 10 & 11 in Part 1 of the Conclusion.

        v.  Save your aligned FASTA file to the desktop of your computer
            so you can proceed with checking your sequences for
            chimeras.

    b.  At the top of the *Greengenes* page you will see a series of
        buttons. Click on the one that says "More Tools".

    c.  The last option is "Chimera Check with Bellerephon". Click it.

    d.  On the Tutorial page navigate to "Chimeras" from either the
        Tutorial main page or the side bar menu.

        i.  Before you proceed with doing the chimera check, please
            first click on the link at the bottom for "What is a
            Chimera?"

        ii. Read through the chimera explanation and then answer
            questions 12 &13 in Part 1 of the Conclusion.

    e.  Work through the steps for chimera checking on the *Greengenes*
        page using the Tutorial to assist you as needed.

    f.  To make sure that your aligned sequences are sent to a valid and
        accessible email address, **use** **your** **school email
        address**.

    g.  We will continue with the next step tomorrow, classifying the
        sequences. For now, once you have submitted your aligned FASTA
        file to be checked for chimeras, do the following:

        1.  Quit the browser you were using (Internet Explorer, Firefox,
            etc.)

        2.  Find the icon for your aligned FASTA file and drag it to the
            trash.

        3.  Shut down the laptop completely -- **WAIT** for the screen
            to go completely black before you close the top!!

        4.  Return the laptop to the cart and check out with the
            teacher.

**Day 3:**

> [Objectives, Day 3]{.underline}:
>
> Students will:
>
> 1\. use the Tutorial to learn about taxonomies.
>
> 2\. use *Greengenes* to classify the sequences.

**[To begin: - (a review)]{.underline}**

i.  Set up with a partner at a computer

ii. Launch a web browser (Firefox, Safari, Internet Explorer)

iii. Open the following web site:
     [http://greengenes.lbl.gov](http://greengenes.lbl.gov/)

iv. On the left hand side there is a menu box titled "About". In that
    box, click on "Tutorial".

v.  Before you proceed with reading today's lesson, first create a split
    screen between the *Greengenes* and Tutorial pages (for **PC**
    users). To do this:

    a.  On the bottom of your screen you can see the windows that are
        active on your desktop. The Tutorial page should be the
        highlighted one.

    b.  Hold down the Ctrl key and click on the *Greengenes* page.

    c.  Now, with both highlighted, right click on one of them and
        select "Tile Vertically". This will split your screen between
        the two pages.

> (**Mac** users can manually resize the windows or use the F9 key to
> bounce between the Greengenes and Tutorial.)

6)  **[Classifying Your Aligned, Clean Sequences]{.underline}**:

    a.  Before you proceed with classification you may want to look at
        the Excel report from *Greengenes*/Bellerephon to see what it
        had to say about your sequences.

        i.  Open your email and find the email returned to you by
            Greengenes after checking for chimeras.

        ii. It will have two files attached. One is an aligned,
            ***clean*** FASTA file. The other is a spreadsheet
            explaining the outcome of the chimeric check process on your
            submitted sequences. Open the spreadsheet (.xls file).

        iii. Were all of the sequences valid or did you have any
             chimeric sequences?

        iv. Save your aligned, ***clean*** FASTA file to the desktop of
            your computer so you can proceed with classifying your
            sequences.

    b.  On the *Greengenes* page go back to the "More Tools" button.
        Click it.

    c.  Select the option that says, "Classify a Batch of Your
        Sequences".

    d.  On the Tutorial page proceed to the next step, "Classification",
        from either the Tutorial main page or the side bar menu.

        i.  In the first paragraph on the Classification page of the
            Tutorial, last line, is a link to find out more about the
            taxonomies used by *Greengenes*. Click it.

        ii. Read through the explanation and comparison of the
            taxonomies and then answer the questions 14 & 15 in Part 1
            of the Conclusion.

    e.  Work through the steps for classifying your sequences on the
        Greengenes page using the Tutorial to assist you, as needed.

    f.  To make sure that your classification report is sent to a valid
        and accessible email address, **use** **your** **school email
        address**.

    g.  Tomorrow we will investigate the classification report that will
        be returned to you by *Greengenes*. For now, once you have
        submitted your aligned, clean FASTA file to be classified, do
        the following:

        1.  Quit the browser you were using (Internet Explorer, Firefox,
            etc.)

        2.  Find the icon for your aligned, clean FASTA file and drag it
            to the trash.

        3.  Shut down the laptop completely -- **WAIT** for the screen
            to go completely black before you close the top!!

        4.  Return the laptop to the cart and check out with the
            teacher.

**Day 4**:

> [Objectives, Day 4]{.underline}
>
> Students will:
>
> 1\. Review the classifications of submitted sequences.
>
> 2\. Compare taxonomies by looking at multiple classifications of the
> same sequence.

7)  **[Comparing Taxonomies]{.underline}**:

    a.  Set up at a computer with your partner.

    b.  Check your email for the classification file returned to you by
        *Greengenes*.

    c.  Save the classification spread sheet to the desktop.

Open the spreadsheet and look it over. There are 31 columns. The first
one identifies the sequence you submitted, the rest are repeating
columns of data, the same five columns of information for each of the
six taxonomies used by *Greengenes*. We are interested in the fifth
column of each taxonomy.

d.  Choose the two taxonomies you would like to compare. G2 chip, Pace,
    Hugenholtz, etc.

e.  Go to the fifth column for one of your chosen taxonomies.

    i.  Double click in the first row of that column.

    ii. You should see the name of the taxonomy followed by the words
        "tax string" -- short for taxonomic string.

    iii. The whole string of classifying information for each sequence
         you submitted is below. If it follows standard taxonomic format
         it will read "Kingdom, Phylum, Class, Order, Family, Genus, &
         species". As you read about taxonomies in the Tutorial, this
         will not be the case for every taxonomy for each sequence.

f.  Identify this list as yours for printing purposes.

    i.  Click to place the cursor right after the words "tax string".

    ii. Type in your name and your partner's name after this. Now you
        will be able to easily identify your list when you print it.

g.  Expand the column so you can read the entire taxonomic string.

    i.  At the top of this column is a letter. Move the cursor to the
        right hand side of this box until the arrow becomes a cross,
        with arrows pointing left and right.

    ii. Clicking and holding the mouse button, drag the right hand edge
        of this box to the right until you can see the entire string for
        each of the sequences.

h.  Print your selection.

    i.  At the top right of your screen is the word "**File**". Click it
        once, and from the drop down menu select "**Page Setup..."**

    ii. When the **Page Setup** box appears, choose "landscape" instead
        of "portrait" as your page orientation, and click "OK". This
        allows you to print the full tax string.

    iii. Back to the top right of your screen and the word "**File**".
         Click it once, and from the drop down menu select "**Print**".

    iv. When the **Print** box shows up, look for and click to choose
        "**selection**". This will print out only the column you just
        highlighted and **not** the entire 31 columns of the
        spreadsheet!!!

i.  Repeat steps f, g, h, and i (above) for the second taxonomy you have
    decided upon.

j.  Retrieve both of your taxonomies from the printer.

k.  Compare the two lists, sequence by sequence. Answer questions 16-18
    in Part 1 of the Conclusion.

l.  Congratulations. You have successfully classified bacteria based on
    a DNA sequence. Before you proceed with Part 2 of the Conclusion,
    please clean up.

    1.  Quit the browser you were using (Internet Explorer, Firefox,
        etc.).

    2.  Find the icon for your classification spreadsheet and drag it to
        the trash.

    3.  Shut down the laptop completely -- **WAIT** for the screen to go
        completely black before you close the top!!

    4.  Return the laptop to the cart and check out with the teacher.

**[Conclusion]{.underline}**:

**Part 1**: On a separate piece of paper, answer each of the following
questions with complete thoughts and in complete sentences.

1.  Name at least three things that a user can do with the *Greengenes*
    web application.

2.  What two pieces of information are contained in a FASTA file?

3.  What are two ways that a user would normally get a FASTA file?

4.  What does the 16S rRNA gene code for?

5.  Give three reasons that researchers use the 16S to identify
    prokaryotes.

6.  What are two main ways that point mutations are created?

7.  What are the three types of point mutations?

8.  Which of the three is least deleterious and why? How does it compare
    to the other two?

9.  What is the relationship between the user sequence and a near
    neighbor or a type strain?

10. Give some similarities between the two files.

11. How are you able to tell which portions of the gene are conserved
    and which are variable? Explain.

12. Historically, what is a chimera? Why do microbiologists use the same
    name for these DNA sequences?

13. Why is it important to have chimeric sequences removed before having
    the sequences classified?

14. Explain two reasons why biologists create different taxonomies (why
    do they disagree!!)?

15. In your own words, what is an OTU?

16. Which two taxonomies are you comparing?

17. Which of the sequences end at Genus and species? Numbered OTU's?

18. Compare the same sequences on both taxonomies. Are they classified
    the same in each taxonomy? Do this for five or six sequences.
    Overall, how do they compare? What general statements can you make
    about how each taxonomy tends to classify?

19. Comparing the two taxonomies, does one seem more useful? Explain.
    Why or why not?

**Part 2**: As a conclusion to this classification activity using DNA
sequences, please answer the following questions. All answers should be
on a separate piece of paper. Answers should be complete thoughts and in
complete sentences.

1)  Explain how the 16S rRNA gene is used in classification of
    prokaryotes. In your answer, include the following:

    a.  Is the entire length of the 16S rRNA gene used equally?

    b.  How and why do taxonomists disagree in their classification of
        prokaryotes using the 16S rRNA gene?

2)  What are the steps that a user of *Greengenes* has to do with their
    sequences before the sequences are classified? For each step,
    include the following:

    a.  What exactly is *Greengenes* doing to the sequence?

    b.  Why is this step important?

3)  Give examples of who would use *Greengenes* professionally. Why
    would these professionals want to classify bacteria and archaea
    (microbial communities)?

4)  Give an example of where/why you might want to explore the microbial
    community? Explain.