Transcript Part 2

Part Two
Identification of bacteria by DNA sequencing
Sequencing Reaction: One Primer, labeled dNTPs, other PCR reagents
Beckman CEQ 8000
Capillary Head Sequencer
Sequences emailed from facility.
FinchTVTM: A free Chromatograph Viewing Program
Each sample: Analyze overall quality of sequence
Example
Chromatogram:
Identify Unknown Bacteria at NCBI:
Basic Local Alignment Search Tool
NCBI: A Worldwide Resource
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Origination of some “HITS” to our sequences
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RESULTS: Monday Lab 2005
Student
Bacteria Source
Primers
NCBI
NCBI % match
Monday
BH
Control
Set One
Bacillus subtilis
98
Monday
PM
Control
Set One
Pseudomonas aeroginosa
98
Monday
PS
Control
Set One
Citrobacter freundii
99
Monday
KY
Control
Set One
Citrobacter freundii
95
Monday
MW
Control
Set One
Enterococcus faecalis
99
Monday
MT
Control
Set One
Enterococcus faecalis
98
Monday
ZZ
Soda Can
Set One
Psuedomonas geroginosa
99
Monday
SN
Soda Can
Set One
Neisserra cinerea
98
Monday
DR
Soda Can
Set One
Brevundimonas vesicularis
99
Monday
JC
Soda Can
Set One
Serratia marcescens
99
Monday
AE
Soda Can
Set One
Klebsiella sp
94
RESULTS: Tuesday Lab 2005
Student
Bacteria Source
Primers
NCBI
NCBI % match
Tuesday
TMM
Control
Set Two
Pseudomonas aeruginosa
96
Tuesday
JM
Control
Set Two
Pseudomonas aeruginosa
96
Tuesday
ES
Control
Set Two
Enterococcus faecalis
99
Tuesday
AM
Soda Can
Set Two
Brevundimonas vesicularis
99
Tuesday
MO
Soda Can
Set Two
Neisseria subflava
99
Tuesday
DA
Soda Can
Set Two
Klebsiella planticola
98
Tuesday
BB
Soda Can
Set Two
Klebsiella sp.
98
Tuesday
BK
Soda Can
Set Two
Neisseria sp.
95
Tuesday
AP
Soda Can
Set Two
Neisseria sp.
99
Tuesday
JS
Soda Can
Set Two
Klebsiella sp.
98
Tuesday
IF
Soda Can
Set Two
Nesseria sp.
97
Tuesday
JW
Soda Can
Set Two
Neisseria sp.
99
RESULTS: Alignment
All Samples Monday Lab
Conserved and Variable Regions
Generated using ClustalW
(free Alignment maker)
RESULTS: Relatedness
Alignments are
the Basis of
Phylogenic
Trees
16SrRNA DNA
amplified using
Primer Set #1
Unrooted tree
(generated by Phylip's Drawtree: another online program)
RESULTS: Generating a longer fragment
The first primer set amplifies the middle of the 16s
DNA, and the second primer set amplifies an end.
The sequences overlap so in the future these
primers could be used to get a longer sequence.
Example Next Slide:
Pseudomonas aeruginosa
Primer Set #1
Pseudomonas aeruginosa isolate: 82% identity match with E. coli
Region of Alignment PCR sample
Full length E. coli 16S rRNA gene
~425 bases of alignable DNA
sequence generated
Primer Set #2
Pseudomonas aeruginosa isolate: 83% identity match with E. coli
Region of Alignment of PCR
Full length E. coli 16S rRNA gene
~280 bases of alignable
DNA sequence generated
Class Results on Spreadsheet
Communicating Science: The Real World
It is just as important to be able to
communicate your results to others as it is to
gather and interpret data.
1) Peer Reviewed Journal Articles
2) Conferences:
a) Short Presentations
b) Poster Sessions
3) Informal Networking, Retreats etc.
Real Deal
2) Talks in General:
Title: Often good to come up with your own
Background:
Attention grabber
What was the question they were trying to answer?
Methods
Compare to those we know
Results: Figures are often the best way to show results
Conclusions…..
Critiques….
What you would do differently
POSITIVE
Future directions………….Applications
2a) Sample Science Talk:
One variant of an Intro to Our Project
Attention
grabber
Methods
Some Key Results….not all
Discussion + Critique
Put in Posters
Are the Bacteria
Dangerous?
Does this change
your view?
2a) Science Talks
2b) Poster sessions:
Adult versions of the Science Fair
Poster session in the parking structure!
Sections for the Poster
TITLE
(Name; Organization)
INTRODUCTION
METHODS
RESULTS
Figures and Tables are good….
Use some we made and
Make some of your own
Gels, Chromatographs, Alignments etc…
Goal: to show a story start to finish in a visual way
CONCLUSIONS / Future Research
Making a Poster
STANDARD WAY: A Giant Power Point Slide
Power Point: under File Menu
Choose “Page Set Up”
Select Dimensions for Poster
Standard Poster Sizes:
Width 48 inches
Height 36 inches
Slides: “landscape”
Or Use a Template (steal from a friend)
Google “poster template” science; or use link on webpage
Often Easiest to:
Put together text in Word or Regular Power
Point size slides
Cut and paste into the giant slide.
Groups for Posters…..
End of semester poster setup
and presentations
Check talk beforehand
Please practice with your
group so everyone knows
their part
Want to Post Your Sequence?
http://www.ncbi.nih.gov/Genbank/index.html
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