# Presentation: 074239

## Outline

-  How to build a phylogenetic tree:
-  Sequence alignment
-  Tree reconstruction
-  How to read a tree
-  ClustalX software
-  clustalx.exe
-  njplotWIN95.exe
-  Phylogenetic marker genes
-  16S rRNA
-  Sequence format
-  FastA
-  Demonstration
-  BLAST search
-  Alignment & tree reconstruction
-  Identification of unknown sequence

- Falk Warnecke, Microbial Ecology Program, JGI, FWarnecke@lbl.gov

**Notes:**
Fairly big project in terms of number of institutes and people involved

## Darwin and Haeckel

**Darwin and Haeckel**

- Haeckel, 1866

- Darwin, 1837

## Darwin and Haeckel

**Darwin and Haeckel**

- Darwin, 1837

## Sequence alignment

- Unaligned:
-             ***     *****
- Sequence A: GTAACGTGATACG
- Sequence B: GTACGTCAATACG

- Conserved positions

**Correct alignment:**
- homologous bases will stand one below the other in a column!

**Notes:**
Fairly big project in terms of number of institutes and people involved

## Sequence alignment

- Unaligned:
-             ***     *****
- Sequence A: GTAACGTGATACG
- Sequence B: GTACGTCAATACG

- Conserved positions

- Aligned:
-             *** *** * ****
- Sequence A: GTAACGTGA-TACG
- Sequence B: GTA-CGTCAATACG

**Correct alignment:**
- homologous bases will stand one below the other in a column!

**Notes:**
Fairly big project in terms of number of institutes and people involved

## Sequence alignment

**Correct alignment:**
- homologous bases will stand one below the other in a column!

- Unaligned:
-             ***     *****
- Sequence A: GTAACGTGATACG
- Sequence B: GTACGTCAATACG

- Conserved positions

- Aligned:
-             *** *** * ****
- Sequence A: GTA**A**CGT**G**A**-**TACG
- Sequence B: GTA**-**CGT**C**A**A**TACG

- Exchanged base / substitution

- Insertion / deletion

**Notes:**
Fairly big project in terms of number of institutes and people involved

## Phylogenetic tree reconstruction

-                   ****
- Sequence A: AAGGTTCCAC
- Sequence B: AAAATTCCAC
- Sequence C: AACCCCCCAC
- Sequence D: GGTTAACCAC
- Sequence E: GGTTGGCCAC

-     A    B    C    D
- A
- B  0.2
- C  0.4  0.4
- D  0.6  0.6  0.6
- E  0.6  0.6  0.6  0.2

**Notes:**
Fairly big project in terms of number of institutes and people involved

## Free software for sequence alignment and tree calculation
Download from: ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX
	(clustalx1.83.zip)

- Download from: ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX
- 	(clustalx1.83.zip)

**ClustalX**

## Phylogenetic marker genes - prerequisites

**16S rRNA**
**23S rRNA**
**Elongation factors**
**EF-Tu**
**EF-G**
**ATP synthase**
***Rec*****A**
***Hsp*****60**
**RNA polymerase**
**Gyrase**

** ****Presence in all organisms**

** ****Functional constancy**

** ****Comprehensive database**

**  ****Complexity**
-  Size (information content)
-  Conserved and variable
-    structure elements

**Phylogenetic marker genes - prerequisites**

## Ribosomal RNA as a phylogenetic marker gene

-  Ubiquitous distribution
-  Functional constancy
-  Large size (information content)
-  Conserved and highly variable structural elements
-  Comprehensive databases available
-  (No lateral gene transfer)
-  Good target for FISH!

**Disadvantages:**
-  No continuous sequence change
-  Multiple genes/operons
-  Different species with identical 16S rRNAs
-  One base change needs nearly one million years

**Ribosomal RNA as a phylogenetic marker gene**

## 16S Ribosomal RNA as a phylogenetic marker gene

## Sequence format: Fasta

- 16S rRNA sequence
- 1441 nt

- >AMD_unknown_sequence
- TCCGGTTGATCCTGCCGGCGGCCACTGCTATCAAGTTCCGACTAAGCCAT
- GCGAGTCAAGGTATCGTAAGATGCCGGCACACTGCTCAGTAACACGTGGA
- TAATCTAACCTTGAGTAAGGGATAACTTCGGGAAACTGAAGGTAATACCT
- TATAATTGCTTAAAACTGGAATGTTTTTGCAATAAAAGTTACGACGCTCA
- AGGATGAGTCTGCGACCTATCAGGTAGTAGGTGGTGTAATGGACCACCTA
- GCCTCAGACGGGTACGGGCCCTGGGAGGGGTAGCCCGGAGATGGACTCTG
- AGACATAAGTCCAGGCCCTACGGGGCGCAGCAGGCGCGAACACTGTGCAA
- TGCGCGAAAGCGCGACACGGGGAGCTTGAGTGTCTTGGCATAGCCAAGAC
- TTTTCTCATTCCTAAAAAGCATGAGGAATAAGTGCTGGGTAAGACGGGTG
- CCAGCCGCCGCGGTAACACCCGCAGCACGAGTAGTGGTCACTTTTATTGA
- GCCTAAAGCGTTCGTAGCCGGTTTTGTAAATCTTCAGATAAAGCCTGAAG
- CTTAACTCCAGAAAGTCTGAAGAGACTGCAAGACTTGAGATCGGGTGAGG
- TTAAACGTACTTTCAGGGTAGGGGTAAAATCCTGTAATCCCGGAAGGACG
- ACCAGTGGCGAAAGCGTTTAACTAGAACGAATCTGACGGTAAGGAACGAA
- GGCTAGGGTAGCAAACCGGATTAGATACCCGGGTAGTCCTAGCTGTAAAC
- ATTGCCCATTTGATGTTGCTTTTCCGTTGAGGGAAGGCAGTGTCGGAGCG
- AAGGTGTTAAATGGGCCGCTTGGGAAGTATGGTCGCAAGACTGAAACTTA
- AAGGAATTGGCGGGGGAGCACCGCAACGGGAGGAATGTGCGGTTTAATTG
- GATTCAACGCCGGAAAACTCACCGGGAACGACCTGTGCATGAGAGTCAAC
- CTGACGAGCTTACTCGATAGCAGGAGAGGTGGTGCATGGCCGTCGTCAGC
- TCGTACCGTAGGGCGTTCACTTAAGTGTGATAACGAGCGAGACCCACATC
- TTTAATTGCAAATGTATATGAGAATATGCATGCACTTTAGAGAAACCGCC
- AGCGCTAAGCTGGAGGAAGGAGTGGTCGACGGCAGGTCAGTACGCCCCGA
- ATTTCCCGGGCTACACGCGCATTACAAAGAACGGGACAATACGTTGCAAC
- CTCGAAAGAGGAAGCTAATCGCGAAACCCGTCCATAGTTAGGATTGAGGG
- CTGTAACTCGCCCTCATGAATCTGGATTCCGTAGTAATCGCGGGTCAACA
- ACCCGCGGTGAACATGCCCCTGCTCCTTGCACACACCGCCCGTCAAACCA
- TCCGAGTTGGTGTTGGATGAGGTTTAATTCGAGAGGGTTAAATCAAATCT
- GATGTCGGTGAGGAGGGTTAAGTCGTAACAAGGTATCCGTA

## Demonstration: Identify unknown organism

-  Starting point: 16S rRNA sequence of an unknown organism
-  Strategy:
- Retrieve closely related reference sequences from Genbank via BLAST
- Compile sequences in Fasta format in one text file
- Do sequence alignment and tree reconstruction using ClustalX
- Identify organism

## Demonstration: 16S rRNA sequence of unknown organism

- >AMD_unknown_sequence
- TCCGGTTGATCCTGCCGGCGGCCACTGCTATCAAGTTCCGACTAAGCCAT
- GCGAGTCAAGGTATCGTAAGATGCCGGCACACTGCTCAGTAACACGTGGA
- TAATCTAACCTTGAGTAAGGGATAACTTCGGGAAACTGAAGGTAATACCT
- TATAATTGCTTAAAACTGGAATGTTTTTGCAATAAAAGTTACGACGCTCA
- AGGATGAGTCTGCGACCTATCAGGTAGTAGGTGGTGTAATGGACCACCTA
- GCCTCAGACGGGTACGGGCCCTGGGAGGGGTAGCCCGGAGATGGACTCTG
- AGACATAAGTCCAGGCCCTACGGGGCGCAGCAGGCGCGAACACTGTGCAA
- TGCGCGAAAGCGCGACACGGGGAGCTTGAGTGTCTTGGCATAGCCAAGAC
- TTTTCTCATTCCTAAAAAGCATGAGGAATAAGTGCTGGGTAAGACGGGTG
- CCAGCCGCCGCGGTAACACCCGCAGCACGAGTAGTGGTCACTTTTATTGA
- GCCTAAAGCGTTCGTAGCCGGTTTTGTAAATCTTCAGATAAAGCCTGAAG
- CTTAACTCCAGAAAGTCTGAAGAGACTGCAAGACTTGAGATCGGGTGAGG
- TTAAACGTACTTTCAGGGTAGGGGTAAAATCCTGTAATCCCGGAAGGACG
- ACCAGTGGCGAAAGCGTTTAACTAGAACGAATCTGACGGTAAGGAACGAA
- GGCTAGGGTAGCAAACCGGATTAGATACCCGGGTAGTCCTAGCTGTAAAC
- ATTGCCCATTTGATGTTGCTTTTCCGTTGAGGGAAGGCAGTGTCGGAGCG
- AAGGTGTTAAATGGGCCGCTTGGGAAGTATGGTCGCAAGACTGAAACTTA
- AAGGAATTGGCGGGGGAGCACCGCAACGGGAGGAATGTGCGGTTTAATTG
- GATTCAACGCCGGAAAACTCACCGGGAACGACCTGTGCATGAGAGTCAAC
- CTGACGAGCTTACTCGATAGCAGGAGAGGTGGTGCATGGCCGTCGTCAGC
- TCGTACCGTAGGGCGTTCACTTAAGTGTGATAACGAGCGAGACCCACATC
- TTTAATTGCAAATGTATATGAGAATATGCATGCACTTTAGAGAAACCGCC
- AGCGCTAAGCTGGAGGAAGGAGTGGTCGACGGCAGGTCAGTACGCCCCGA
- ATTTCCCGGGCTACACGCGCATTACAAAGAACGGGACAATACGTTGCAAC
- CTCGAAAGAGGAAGCTAATCGCGAAACCCGTCCATAGTTAGGATTGAGGG
- CTGTAACTCGCCCTCATGAATCTGGATTCCGTAGTAATCGCGGGTCAACA
- ACCCGCGGTGAACATGCCCCTGCTCCTTGCACACACCGCCCGTCAAACCA
- TCCGAGTTGGTGTTGGATGAGGTTTAATTCGAGAGGGTTAAATCAAATCT
- GATGTCGGTGAGGAGGGTTAAGTCGTAACAAGGTATCCGTA

## Demonstration: BLAST search for closely related reference sequences

## Demonstration: download and compile text file

- >AMD_unknown_sequence
- TCCGGTTGATCCTGCCGGCGGCCACTGCTATCAAGTTCCGACTAAGCCAT
- ...
- >Ferroplasma_acidiphilum
- CTCGCTCGCCCATCYGGTTGATCCTGCCGGCGGCCACTGCTATCAAGTTC
- ...
- >Ferroplasma_cyprexacervatum
- TTCTGGTTNGATCCTGCCGGGCGGCCACTGCTATCAAGTTCCGACTAAGC
- ...
- >Thermoplasma_volcanium
- CGGTCACTGCTATCAGGTTCCGACTAAGCCATGCAAGTCACGGGGCCGTA
- ...
- >Picrophilus_oshimae
- ATTCTGGTTGATCCCGGCGGCGGCCACTGCTATCAAGTTCCGACTAAGCC
- ...
- >AMD_F.acidarmanus_TypI
- TCCGGTTGATCCTGCCGGCGGCCACTGCTATCAAGTTCCGACTAAGCCAT
- ...
- >AMD_F.acidarmanus_TypII
- TCCGGTTGATCCTGCCGGCGGCCACCGCTATCAAGTTCCGACTAAGCCAT
- ...
- >AMD_Thermoplasmatales
- GGTTGATCCTGCCGGCGGCTACTGCTATCAGGTTTCGACTAAGCCATGCG
- ...

## Demonstration: sequence alignment and tree reconstruction