1. Workshop on the analysis of microbial sequence data using ARB
August , 2009 University of Hawaii Instructors: Amy Apprill, UH & WHOI Megan Huggett, UH
Sponsored by:

2. By the end of the workshop you will be able to:
Efficiently work with raw nucleotide sequence data and prepare sequences for upstream analysis
Be familiar with the ARB software package
Align sequences using ARB
Construct phylogenetic trees using ARB
Utilize a few of the web-based resources available for sequence analysis & phylogenetics
Design probes & primers using ARB
Construct a functional gene database using ARB

3. Workshop schedule Thursday, August 13:
9:00 – 10:00am: Overview of workshop, Lecture: Introduction to the theory of
phylogenetic analyses and the ARB program
10:00 – 10:40am: Tutorial 1 – Clean-up, assemblage and quality control of sequence data
10:40 – 11:00am: Coffee break
11:00 – 11:15am: Lecture: Sequence Alignments
11:15 – 12:30pm: Tutorial 2 - Importing and aligning sequences into an ARB database
12:30 – 1:30pm: Lunch break – Discussion: Student introductions
1:30 – 2:00pm: Free time to complete tutorial 2
2:00 – 2:30pm: Lecture: Overview of phylogenetic comparisons and tree-building
2:30 – 3:40pm: Tutorial 3 – Creating a phylogenetic comparison of the newly imported and aligned sequences
3:40 – 4:00pm: Afternoon break
4:00 – 4:30pm: Tutorial 4 – Creating a sequence alignment filter
4:30 – 5:00pm: Tutorial 5 – Using BLAST to identify related sequences

4. Workshop schedule Friday, August 14:
9:00 – 9:30am: Recap of day 1, intro to assignment; question & answer session
 9:30 – 10:40am: Free time to complete tutorials & assignment
10:40 – 11:00am: Coffee break
11:00 – 12:00pm: Free time to complete assignment
12:00 – 1:00pm: Lunch break –
Discussion: Opportunities in science education for PhD’s
 1:00 – 2:00pm: Free time to complete assignment, question and answer session
 2:00 – 2:30pm: Lecture: Primer and probe design
 2:30 – 3:40pm: Tutorial 6: Primer and probe design
 3:40 – 4:00pm: Afternoon break
 4:00 – 5:00pm: Tutorial 7: Creating and working with new databases

5. Lecture 1: Introduction to the theory of microbial phylogenetics & the ARB program

6. Phylogeny in microbiology
Morphology
Requires cultured isolates (e.g. Petri dishes)
Limited information content
Chemical cell components (phospholipids, fatty acids)
Usually misses 99% of diversity because:
Culture conditions can’t be copied
Many species can’t be exist in isolation

7. Phylogeny in microbiology
The alternative
Using molecular tools to uncover previously unknown microbial diversity
16S/18S rRNA has become the standard molecular marker
Of course, we still need isolates!
Results from microbial genome and metagenome sequencing can particularly guide isolation studies
Sequence of molecular marker genes - High information content, low cost, limited labor

8. What makes a good phylogenetic marker gene?
Presence in all organisms
Functional constancy
Complexity
Size
Conserved and variable structural elements
Comprehensive database
Some examples:
16S rRNA, 23S rRNA, ATP synthase, RNA polymerase….

9. Ribosomal RNA as a phylogenetic marker
 Small subunit (SSU) or 16S ribosomal RNA gene
16s rRNA gene
Genomic DNA
Transcription
Folding
rRNA
Becomes part of
the ribosome,
vital for protein synthesis

10. Ribosomal RNA as a phylogenetic marker
Advantages:
Ubiquitous distribution
Regions of high variability
Constant rate of evolution
Naturally replicated in situ
Conserved & variable regions
Large databases available
Conserved PCR primer sets for genes

11. Ribosomal RNA as a phylogenetic marker
Disadvantages:
Many copies per genome (microheterogeneity)
Limited information (only 1500bp, relatively few of which are informative)
Different species have identical 16S rRNAs

12. Secondary structure of ribosomal RNA: constrained mutation across 2-3 billion years
Escherichia
(Bacteria)
Methanococcus
(Archaea)
Saccharomyces
(Eukarya)

13. Secondary structure of ribosomal RNA
Complex pattern of short double-stranded stems, unpaired single-stranded loops and bubbles

14. Secondary structure of ribosomal RNA
Eucarya
Bacteria
Archea
Conserved

15. Modern tree of life based on 16S/18S rRNA sequences

16. Methods to analyze rRNA
Environmental sample
DNA extraction
Detection of cells
PCR amplification
Fluorescence in situ hybridization
Cloning & sequencing
Probe design
Sequence analysis
Sequence database

17. Phylogenetic sequence analysis - the bad news
Phylogenetic analysis needs:
Good sequence data
Proper alignment, analysis and statistical testing
Tidy presentation
“Probes are only as good as the data behind them”
Phil Hugenholtz (1988)
Good data are critical

18. Phylogenetic sequence analysis - the good news
Arb can help you:
Organize and collect your data
Align your data
Make trees
Design probes

19. Phylogenetic sequence analysis steps
Sequence determination
Preparation of raw sequence data (removal of cloning vector, clean ends, quality control, assemble contigs)
Import sequences into database
Alignment of sequences to secondary structure
Phylogenetic comparison

20. Gene cloning & sequencing
TATGCTTGCAA
TTGGTTCT
ACGCTGCT
ATTGCCTA
DNA
PCR amplify gene
Clone DNA using
E. Coli & plasmid insert
ATTGCCTA
TTGGTTCT
TATTGCCTT
TTTGCCTT
ATTGCCTT
Sequence

21. Cloning vector

22. Cloning vector

23. Sequence analysis software – Sequencher (Mac or PC)

27. SSU rRNA gene databases
Silva rRNA based database project (
Well maintained SSU & LSU databases available
Most recent SSU database contains 368,368 quality filtered sequences!
Living Tree Project databases (typed species)
Sequence alignment function (SINA)
Green genes (
SSU rRNA gene database
Sequence trimming functions
Alignment functions
Probe & primer design functions

28. ARB Tutorials Importing sequences into ARB
Aligning sequences using ARB & SINA web-based aligner
Adding sequences to ARB database parsimony tree
Creating sequence filters
Importing sequences from GenBank into ARB
Constructing a phylogeny of sequences
Probe & primer design
Constructing databases for functional genes

29. Tutorial 1 objectives Become familiar with sequence chromatograms
Trim cloning vector sequence files
Trim the ends of sequences
Assemble sequence fragments into contiguous sequences
Export the sequences into a format for ARB