Topics in Bioinformatics 21224 BINF 6010

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Topics in Bioinformatics - 21224 - BINF 6010
Survey of Metagenomics Cameron Applied Research
Center 274 Tues, Thus - 200 pm - 330 pm
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Class mechanics What is metagenomics?
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You can give them a fake e-mail address if you
want to!
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Goals of the class To develop your skill in
critically reading and presenting primary
literature. For you to do an independent
project in an area that you are interested
in. To learn about a rapidly changing area of
biology and bioinformatics.
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On Feb 17-19, brief (non-binding) discussion of
your proposed independent project,
There will be many suggested topics presented
during the early part of the semester
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There will be one exam. The exam is likely the
easiest part of the class!
The study guide has sample questions. Some (but
not all) of the questions on the exam will be
drawn from the sample questions.
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Depending on how many people take the class, you
will do 1 or 2 presentation of papers in the
literature. We do this in the second half of the
semester so that it can be a paper that you need
to know for your independent project
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At the end of the semester. Presentations on the
independent project and a write-up.
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My last seminar class (on microarray
analysis) had independent projects that yielded 4
papers..
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Depending on the of students taking the class,
and how things are going, the schedule and due
dates can (and will) change. Check your e-mail
regularly!
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Class mechanics What is metagenomics?
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Microbes run the world.
Microbes help determine nutrient and energy
flux in our bodies
Microbes help determine nutrient and energy
flux in our planet
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In the 19th century, Pasteur (and others)
demonstrated some of the properties of microbes
Famous demonstration that microbial life is not
spontaneously generated
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Even 20 years ago, we could only study microbes
one at a time, in culture, in the lab away from
their natural environments
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DNA sequencing (and other techniques) is changing
that
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In pure culture, you are looking at one genome at
a time. When you sample microbes in their native
environment, you are looking at many microbes at
a time. Many genomes in a single experiment
Metagenomics
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Three questions that are the focus of
metagenomics 1. Who (which taxa) are there? 2.
What are they doing? 3. What can they do for
us? Lets start with question 1.
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A state-of-the art metagenomics experiment 15
years ago
Some microbial community that you are interested
in (for example the human gut)
Isolate microbial DNA from this community
The DNA isolation doesnt give us enough DNA to
work with
Amplify DNA using PCR (the polymerase chain
reaction)
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http//en.wikipedia.org/wiki/Polymerase_chain_reac
tion
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Some key features of PCR You have to know the
beginning and end of the sequence you want to
amplify ( or you can use random primers) Very
small quantities of template DNA can be used to
generate on the order of 100s of nanograms of
DNA. The trick is thermocycling. Raising the
temperature high enough to denature
double-stranded DNA. Originally, these high
temperatures denatured the polymerase (the enzyme
that synthesizes the DNA). But then Taq was
discovered in hot springs at Yellowstone (a good
example of the utility of microbes in
their native environment). There is a limit on
the length of the amplified DNA (or the
amplicon) that is on the order of 1000s of
nucleotides
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PCR and TAQ are key to the biotechnology
revolution that starts in the late 80s
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A state-of-the art metagenomics experiment 15
years ago
Some microbial community that you are interested
in (for example the human gut)
Isolate microbial DNA from this community
The DNA isolation doesnt give us enough DNA to
work with
Amplify DNA using PCR (the polymerase chain
reaction)
So what do we do with our amplified metagenomic
DNA??
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By selecting the primers, we can target a
specific gene. The most commonly targeted gene
for microbes is the 16S rRNA gene.
Ribosomal rRNA is involved in making
proteins. Among the most conserved genes in
microbial genomes A barcode gene. The
argument (10 years ago) was that we could use
this one gene as a barcode to see which taxa we
were dealing with? Answer the question who is
there?
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we will be getting to know well the 16S rRNA gene
well over the course of this semester!
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The RDP (Ribosomal Database Project) has
somewhere on the order of three quarters of a
million of these 16S rRNA sequences
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In this paper,which we will cover later in the
semester, there are 900,000 16S rRNA sequences
from a single study!
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Data glut Drinking from the firehouse Why this
class (and my department) exists!
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The 16S rRNA gene has conserved and variable
regions. The 900,000 sequences are short reads
from the V3 and V6 regions (much, much more about
this later)
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For those on the computational end of things,
there are many possible independent projects
concerning how much it matters where you place
primers and how you analyze the
resulting sequences Much more about this in the
next few lectures
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But millions of sequences is today, lets go back
again 15 years in time
Some microbial community that you are interested
in (for example the human gut)
Isolate microbial DNA from this community
The DNA isolation doesnt give us enough DNA to
work with
Amplify DNA using PCR (the polymerase chain
reaction)
So what do we do with our amplified metagenomic
DNA??
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In a typical PCR experiment
A gel is a porous semi-solid material with lots
of small holes. DNA is negatively charged.
You place a positive charge on one end of the gel
and the DNA on the other end of the gel and the
DNA migrates towards the charge. Smaller DNA
goes faster. So this agarose gel separates
DNA based on size.
This isnt going to work to well for a 16S rRNA
survey because most of the sequences are
approximately the same length. So if you ran a
gel, you would just see one band!
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State of the art 1993
Denaturing Gradient Gel Electrophoresis (DGGE)
http//en.wikipedia.org/wiki/Temperature_gradient_
gel_electrophoresis
You have a UREA gradient running down the gel.
High concentrations of UREA At one end of the
gel and low concentrations at the other end.
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DGGE State of the art - 1993
You have a urea gradient running down the gel.
High concentrations of urea At one end of the
gel and low concentrations at the other end.
Sequences with high GC content separate at a
different urea concentration than sequences with
low concentration. You get distinct patterns of
bands from different 16S sequences. (You dont
know what the taxa are)
My lab did some DGGE when we were first starting
metagenomics
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Angelo Coast Range Reserve Natural History
24 km from Pacific Ocean Elevation ranges
378-1290 m Preserve is high in the Coast Range
mountains Mainstem and tributaries support
salmon populations
Mary Power
http//angelo.berkeley.edu/Photographs/bio1b/bio1b
.htm
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My lab did some DGGE when we were first starting
metagenomics. (Todd Steck in biology has a setup
that I think is still sitting in my lab)
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The microbial community has been treated as a
black box. We want to look in the black box.
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Advantages of DGGE Very inexpensive. Otherwise,
I cant really think of any. Disadvantages of
DGGE The thin acrylimide gel is easy to break
and hard to pour. Low resolution view. We dont
know which bands correspond to which
sequences The gel can bend making image analysis
difficult.
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The Mallard Creek Wastewater treatment facility
in Charlotte, NC
Helene Hilger
Microbes in this wastewater treatment plant are a
crucial tool in environmental protection
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Next time We will progress beyond 1993! Read
the summary and Chapter 1 in the New Science
of Metagenomics book.